p16-Rb-HDAC1-E2F1对胆道癌的生长作用及其蛋白信号传导通路研究
摘要
肿瘤的发生是一个多因素、多步骤的过程，涉及染色体畸变、抑癌基因的失活、癌基因的活化、细胞周期的调控、基因启动子的转录活化和表遗传学等诸多方面。而对肿瘤的研究也广泛开展在这些纷繁芜杂的方面中。随着人类基因组测序工作完成，人类已进入后基因组时代，基因调控即遗传信息是如何精确调控和准确表达的成为当今的重点研究方向。而遗传信息的调控和表达是在蛋白信号的传导和作用下完成的，所以对蛋白质的研究成为新的研究热点。
在邹声泉教授的领导下，我们的课题组在前期的研究中发现抑癌基因p16和组蛋白乙酰化酶HDAC1与肝外胆管癌的细胞分子遗传学和表遗传学有关联，提示其在肝外胆管癌的发生和发展中扮演着重要角色。另有研究认为，Rb／E2F1途径是细胞周期调控的中心环节并可以调节HDAC1的活性进而影响DNA乙酰化，Rh／p16途径在肿瘤中的作用也逐渐得到公认。这些研究结果提示，p16、Rb、HDAC1和E2F1之间可能在胆道肿瘤中存在一个具有相互反馈调节功能的蛋白信号传导通路。这些蛋白信号涉及抑癌基因的失活、组蛋白乙酰化、基因启动子转录活化和肿瘤表遗传学等诸多方面，所以其在肿瘤演变过程中极具有代表性。
为此本课题从研究肝外胆管癌和胆囊癌p16、Rh、HDAC1和E2F1之间的蛋白信号传导着手，应用质粒转染、免疫沉淀、Western blot、RT-PCR、MTT和免疫组化等实验方法，从细胞水平到组织水平，从体外作用到体内作用，多角度、多层面的研究这些蛋白信号的传导途径及其蛋白质之间的相互作用，探讨这些蛋白的功能及其与肿瘤的基因调控和表遗传学等方面的关系，期望能够为阐明其在胆道癌中的作用机制提供新的理论基础和实验依据。同时将这些蛋白质的表达特征与胆道癌的临床流行病学相联系，期望能从众多纷繁的临床特征中找到与这些蛋白质相关联的因素，从而为寻找胆道癌的病因提供新的证据。但由于时间的关系，本课题尚局限于较浅的层面，在今后的工作中我们将进一步深入研究。
第一部分胆道癌p16-Rb-HDAC1-E2F1中各种蛋白质之间的相互作用及其信号传导途径的研究
论文1 p16-Rb-HDAC1-E2F1在胆道癌细胞系中的表达
目的研究p16-Rb-HDAC1-E2F1中各种蛋白及其mRNA在肝外胆管癌和胆囊癌细胞系中的表达情况。
方法用免疫组织化学和RT-PCR方法检测肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)中p16-Rb-HDAC1-E2F1蛋白及其mRNA的表达。
结果
1．P16 mRNA在QBC_(939)细胞有弱表达，在KMBC细胞、Mz-ChA-1细胞和OZ细胞无表达；Rb mRNA在KMBC细胞有弱表达，在QBC_(939)细胞、Mz-ChA-1细胞和OZ细胞无表达；HDAC1 mRNA在QBC_(939)细胞、KMBC细胞、OZ细胞和Mz-ChA-1细胞均有弱表达；E2F1 mRNA在QBC_(939)细胞、KMBC细胞和Mz-ChA-1细胞有弱表达，在OZ细胞无表达。p16蛋白在QBC_(939)细胞有表达；Rb蛋白在KMBC细胞有表达；HDAC1蛋白在QBC_(939)细胞、KMBC细胞、OZ细胞和Mz-ChA-1细胞均有表达；E2F1蛋白在QBC_(939)细胞、KMBC细胞和Mz-ChA-1细胞有表达。
2．P16蛋白在QBC_(939)细胞种植瘤中有弱表达；Rb蛋白在KMBC细胞种植瘤中有弱表达；HDAC1蛋白在QBC_(939)、KMBC、OZ和Mz-ChA-1细胞种植瘤中有表达，但表达丰度在同种细胞种植瘤中不同；E2F1蛋白在QBC_(939)、KMBC和Mz-ChA-1细胞种植瘤中有弱表达。
结论P16、Rb、HDAC1、E2F1参与了胆道癌的进程；体内环境可对胆道癌细胞系的生物学特性产生影响。
论文2胆道癌p16-Rb-HDAC1-E2F1中各种蛋白质之间的相互作用研究
目的研究胆道癌p16-Rb-HDAC1-E2F1中各种蛋白质之间的相互作用及其机理。
方法用免疫沉淀法检测分别转染p16质粒和pRb质粒的肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)的p16-Rb-HDAC1-E2F1中各种蛋白质之间的相互作用。
结果
1．用HDAC1抗体免疫沉淀下来的蛋白中有Rb蛋白(在QBC_(939)、KMBC、OZ和Mz-ChA-1细胞系)和E2F1蛋白(在QBC_(939)、KMBC和Mz-ChA-1细胞系)的存在，无p16蛋白存在。
2．用Rh抗体免疫沉淀下来的蛋白中有HDAC1蛋白(在QBC_(939)、KMBC、OZ和Mz-ChA-1细胞系)和E2F1蛋白(在QBC_(939)、KMBC和Mz-ChA-1细胞系)的存在，无p16蛋白存在。
结论
1．胆道癌Rb蛋白、HDAC1蛋白和E2F1蛋白之间在细胞水平存在特异的相互作用，表明其蛋白信号之间存在直接的传导途径。
2．P16蛋白在胆道癌中与Rb蛋白、HDAC1蛋白和E2F1蛋白之间并不存在直接的相互作用。
第二部分p16-Rb-HDAC1-E2F1蛋白与胆道癌生物学特性的关系
论文3 p16-Rb-HDAC1-E2F1蛋白在胆道癌细胞中的作用及其相互影响
目的研究p16-Rb-HDAC1-E2F1在体内、体外对肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)生长的作用及其各蛋白之间的相互影响。
方法
1．转染p16质粒和pRh质粒进入肝外胆管癌细胞系(QBC_(939)、KMBC、OZ celllines)和胆囊癌细胞系(Mz-ChA-1 cell line)，用MTT法检测转染前后各胆道癌细胞系生长的变化，用RT-PCR法检测转染p16质粒前后各胆道癌细胞系Rb mRNA的表达变化和转染pRb质粒前后各胆道癌细胞系p16 mRNA的表达变化。
2．用与HDAC1功能相反的去乙酰化酶抑制剂-曲古抑菌素(trichostatinA，TSA)作用于肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)，用MTT法检测TSA对这些胆道癌细胞系生长的影响。
3．分别将转染p16质粒、pRb质粒和经TSA处理的胆囊癌细胞系(Mz-ChA-1cell line)接种裸鼠皮下建立胆囊癌裸鼠种植模型，观察其转染p16质粒和pRb质粒前后和TSA处理前后裸鼠体内种植瘤生长的变化。
结果
1．转染p16质粒的KMBC细胞的Rb mRNA的表达受到抑制。转染pRb质粒的OZ和Mz-ChA-1细胞的p16 mRNA出现弱表达，QBC_(939)细胞和KMBC细胞的p16mRNA的表达无变化。
论文4 HDAC1与凋亡在胆囊癌中的相关性及其在免疫逃逸机制中的作用研究
目的
1．研究凋亡的死亡信号受体途径(Fas／FasL途径)和组蛋白去乙酰化酶HDAC1在胆囊癌生物学行为中的作用。
2．探讨HDAC1在胆囊癌免疫逃逸机制中的相关作用和机制。
方法
1．应用免疫组织化学方法研究胆囊癌组织、胆囊腺瘤组织、胆囊上皮不典型增生组织和慢性胆囊炎组织中Fas／FasL蛋白的表达和HDAC1蛋白的表达情况。
2．运用TUNEL法检测在胆囊癌组织、胆囊腺瘤组织、胆囊上皮不典型增生组织和慢性胆囊炎组织中的原位组织细胞的凋亡情况及其浸润的淋巴细胞的凋亡情况。
3．比较在不同的胆囊癌临床病理类型中Fas蛋白和FasL蛋白的表达情况的差异和在原位胆囊癌组织浸润的淋巴细胞的凋亡情况的差异。
4．探讨胆囊癌Fas／FasL蛋白的表达与组蛋白去乙酰化酶HDAC1蛋白的表达的相关性。
结果
1．Fas蛋白表达的阳性率在胆囊癌组织、胆囊腺瘤组织、胆囊上皮不典型增生组织和慢性胆囊炎组织之间的差异无显著意义。FasL蛋白表达的阳性率在胆囊癌组织显著高于慢性胆囊炎组织(X~2=4.89，P＜0.05)。
2．胆囊癌的组织细胞的凋亡指数显著高于胆囊腺瘤的组织细胞(t′=4.19，P＜0.01)和慢性胆囊炎的组织细胞(t′=8.06，P＜0.01)的凋亡指数；高分化的胆囊癌的组织细胞的凋亡指数显著低于低分化的胆囊癌的组织细胞的凋亡指数(t′=2.63，P＜0.05)，NevinⅠ、Ⅱ、Ⅲ期的胆囊癌的组织细胞的凋亡指数显著低于NevinⅣ、Ⅴ期的胆囊癌的组织细胞的凋亡指数(t′=3.33，P＜0.01)。
3．浸润的淋巴细胞的数量在胆囊腺瘤组织显著低于在胆囊癌组织(t′=6.99，P＜0.01)，在慢性胆囊炎组织显著低于在胆囊腺瘤组织(t′=3.66，P＜0.01)；在高分化胆囊癌组织显著低于在低分化胆囊癌组织(t′=5.31，P＜0.01)，在胆囊癌NevinⅠ、Ⅱ、Ⅲ期组织显著低于在胆囊癌NevinⅣ、Ⅴ期组织(t=-3.76，P＜0.01)。
4．浸润的淋巴细胞的凋亡数量在高分化的胆囊癌组织显著低于在低分化的胆囊癌组织(t=-2.52，P＜0.05)；在NevinⅠ、Ⅱ、Ⅲ期的胆囊癌组织中浸润的淋巴细胞的凋亡数量与在NevinⅣ、Ⅴ期的胆囊癌组织浸润的淋巴细胞的凋亡数量之间的差异无显著意义(t=-1.42，P＞0.05)；在胆囊腺瘤组织中和慢性胆囊炎组织中未见到浸润的淋巴细胞的凋亡。
5．HDAC1蛋白和FasL蛋白在胆囊癌组织的表达呈显著正相关(P＜0.05)，HDAC1蛋白和Fas蛋白在胆囊癌组织的表达无显著相关性。
结论
1．胆囊癌组织细胞表达的FasL通过诱导在癌组织浸润的淋巴细胞的凋亡从而使癌细胞逃避机体的免疫监视，它的上调表达在胆囊癌的分化、浸润和转移过程中扮演着重要角色。
2．未证实胆囊癌组织细胞表达的Fas蛋白在胆囊癌的免疫逃逸中具有作用。
3．组蛋白去乙酰化酶HDAC1蛋白和参与胆囊癌免疫逃逸的FasL蛋白在胆囊癌组织的表达呈显著正相关，提示我们HDAC1与胆囊癌的免疫逃逸具有一定的相关性。
第三部分组蛋白去乙酰化酶抑制剂对胆道癌p16-Rb-HDAC1-E2F1蛋白信号传导的影响
论文5组蛋白去乙酰化酶抑制剂对肝外胆管癌细胞和胆囊癌细胞p16-Rb-HDAC1-E2F1蛋白信号传导的影响
目的研究组蛋白去乙酰化酶抑制剂-曲古抑菌素(trichostatin A，TSA)对肝外胆管癌和胆囊癌p16-Rh-HDAC1-E2F1蛋白信号传导的影响。
方法用TSA作用于肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)，然后用RT-PCR检测p16、Rb、HDAC1、E2F1之mRNA的表达变化，用Western blot检测其蛋白的表达变化。将这些细胞接种在裸鼠皮下建立肝外胆管癌和胆囊癌裸鼠种植瘤模型，用免疫组织化学方法观察TSA在体内对裸鼠种植瘤组织中Rb、HDAC1、DNMT1、E2F1蛋白表达的影响。
结果
1．TSA可以减弱肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lities)和胆囊癌细胞系(Mz-ChA-1 cell line)HDAC1 mRNA和蛋白的表达；增强KMBC细胞系p16 mRNA和蛋白的表达；对各细胞系Rh和E2F1 mRNA和蛋白的表达无影响。
2．TSA作用前后的肝外胆管癌细胞系和胆囊癌细胞系裸鼠成瘤组织中的各种蛋白的表达均无变化。
结论TSA可以抑制肝外胆管癌和胆囊癌HDAC1的表达，并在Rb阳性的细胞中促进p16的表达，从而进一步影响遗传信息的转录。TSA的作用存在时效性。
第四部分p16-Rb-HDAC1-E2F1蛋白与胆囊癌临床病理学的关系
论文6 p16-Rb-HDAC1-E2F1蛋白与胆囊癌临床病理学因素的关系分析
目的探讨胆囊癌的临床病理学特征并研究p16-Rb-HDAC1-E2F1蛋白与胆囊癌临床病理学因素的关系及其意义。
方法
1．回顾性分析华中科技大学附属同济医院1991-2005年收治的65例胆囊癌病例。
2．应用免疫组化法研究胆囊痛组织中p16-Rb-HDAC1-E2F1蛋白的表达水平及特征。
结果
1．本组胆囊癌合并胆囊结石的比例为44.64％(25／56)。胆囊癌患者有乙肝病史的比例为26.79％(15／56)，有血吸虫疫水接触史的比例为30.36％(17／56)。多产者比例为72.98％(27／37)。本组胆囊癌病例占同期胆囊切除术总数的0.64％(56／8807)。术前误诊疾病依次为结石、腺瘤和肝癌。影像学检查中，B超的正确率为42.3％(22／52)，CT的正确率为57.1％[(14-6)／(20-6)]，MRI的正确率为50％[(5-2)／(8-2)]。有4例胆囊癌患者检查了血γ-GT，其结果均高于正常值。
2．本组胆囊癌原发部位多位于胆囊颈部(29.73％，11／37)和体部(29.73％，11／37)，其次为胆囊底部(16.22％，6／37)，位于胆囊管部占5.41％(2／37)，位于胆囊体部和底部占13.51％(5／37)，另有5.41％(2／37)位于胆囊颈体交界部。胆囊癌组织学分型依次为腺瘤(83.93％，47／56)、腺瘤恶变(7.14％，4／56)、鳞癌(5.36％，3／56)和未分化癌(3.57％，2／56)。本组胆囊癌Nevin分期，Ⅰ期占14.29％(8／56)，Ⅱ期占10.71％(6／56)，Ⅲ期占26.79％(15／56)，Ⅳ期占14.29％(8／56)。胆囊癌分级，高分化占44.64％(25／56)，中分化占12.5％(7／56)，低分化占42.86％(24／56)。本组病例的胆囊癌的原发部位与肿瘤的分级、分期和患者性别均无显著相关性。胆囊癌转移部位多为肝脏(14／56)。有2例肿瘤组织浸润胆囊壁内神经束衣。
3．P16蛋白和Rb蛋白在胆囊癌组织中低表达，HDAC1蛋白和E2F1蛋白在胆囊癌组织中高表达，其表达均与胆囊癌的分化程度、Nevin分期、患者的年龄、性别和是否合并结石无显著相关性。P16蛋白在胆囊癌的表达率在合并乙肝者为27.7％，在没有合并乙肝者为55.6％，二者之间的差异具有显著性意义(P＜0.05)；Rb蛋白、HDAC1蛋白和E2F1蛋白在胆囊癌的表达均与患者是否合并乙肝无显著相关性。P16蛋白和Rb蛋白在胆囊癌的表达呈显著正相关(γ=0.6091，t=4.48，P＜0.001)，p16蛋白和HDAC1蛋白在胆囊癌的表达呈显著负相关(γ=-0.5215，t=3.56，P＜0.005)，Rb蛋白和HDAC1蛋白在胆囊癌的表达呈显著负相关(γ=-0.3953，t=2.51，P＜0.05)，E2F1蛋白和其它蛋白在胆囊癌的表达无显著相关性。
结论
1．胆囊结石、乙肝病毒感染、血吸虫感染和多产妇是胆囊癌的高危因素；γ-GT可能是胆囊癌有潜力的肿瘤标志物。
2．胆囊癌多位于胆囊颈部和体部，组织分型以腺癌居多，并具有沿神经浸润的特性；胆囊癌的恶性程度与其原发部位和患者性别无关。
3．P16蛋白、Rb蛋白、HDAC1蛋白和E2F1蛋白在胆囊癌的表达均与肿瘤的恶性程度、患者的年龄和性别无显著相关性。
4．P16蛋白在胆囊癌的表达与患者是否合并乙肝具有显著相关性。
5．P16蛋白、Rb蛋白的低表达和HDAC1蛋白、E2F1蛋白的高表达参与了胆囊癌的进程，并且p16蛋白、Rb蛋白和HDAC1蛋白在胆囊癌中具有相互协同作用。
目的探讨意外胆囊癌的临床病理学特征并研究p16-Rb-HDAC1-E2F1蛋白与意外胆囊癌临床病理学因素的关系及其意义。
方法回顾性研究同济医院23例意外胆囊癌病例，并和同期收治的33例术前确诊的胆囊癌病例进行比较分析，并应用免疫组化法研究意外胆囊癌组织中p16-Rb-HDAC1-E2F1蛋白的表达水平及特征。
结果意外胆囊癌合并胆囊结石的比例高于术前确诊的胆囊癌，差异有非常显著意义(P＜0.01)。患者有乙肝病史的比例在意外胆囊癌为21.74％(5／23)，在术前确诊的胆囊癌为30.30％(10／33)。患者有血吸虫疫水接触史的比例在意外胆囊癌为39.13％(9／23)，在术前确诊的胆囊癌为24.24％(8／33)。多产(大于三胎)的比例在意外胆囊癌为56.52％(13／23)，在术前确诊的胆囊癌为42.42％(14／33)。分别有2例意外胆囊癌和2例术前确诊的胆囊癌检查了血．γ-GT，其结果均高于正常值。意外胆囊癌的原发部位多位于胆囊颈部和体部，术前确诊的胆囊癌原发部位多位于胆囊体部和底部。意外胆囊癌的Nevin分期，在Ⅰ期和Ⅱ期的比例均高于术前确诊的胆囊癌，差异有显著意义(P＜0.05)；在Ⅳ期的比例低于术前确诊的胆囊癌，差异有显著意义(P＜0.05)；在Ⅴ期的比例低于术前确诊的胆囊癌，差异有非常显著意义(P＜0.01)。意外胆囊癌的分级，高分化者高于术前确诊的胆囊癌，差异有显著意义(P＜0.05)；低分化者低于术前确诊的胆囊癌，差异有显著意义(P＜0.05)。p16-Rb-HDAC1-E2F1蛋白的表达在意外胆囊癌和非意外胆囊癌的差异无显著性意义。
结论意外胆囊癌具有肿瘤原发部位以胆囊颈、体部居多、恶性程度较术前确诊的胆囊癌低和结石的并存率较术前确诊的胆囊癌高的特征；结石、乙肝、血吸虫和多产是意外胆囊癌的高危因素。意外胆囊癌和非意外胆囊癌具有相似的分子生物学特征。
本课题从研究蛋白质的功能着手，探讨了肝外胆管癌和胆囊癌p16-Rb-HDAC1-E2F1之间的蛋白信号传导途径及其蛋白质之间的相互作用，得出以下结论和创新点：
1．胆道癌p16-Rb-HDAC1-E2F1之间的蛋白信号传导涉及抑癌基因的失活、细胞周期的调控、组蛋白乙酰化、基因启动子的转录活化和肿瘤表遗传学等几个方面，本课题将它们结合起来进行研究，更有代表性的说明胆道癌演变过程的复杂性。
2．胆道癌Rb蛋白、HDAC1蛋白和E2F1蛋白之间在细胞水平存在特异的相互作用，表明其蛋白信号之间存在直接的传导途径。p16蛋白在胆道癌中与Rb蛋白、HDAC1蛋白和E2F1蛋白之间存在间接的相互作用，并且证实p16对胆道癌的抑制作用依赖于Rb的功能。提示在胆道癌中存在一个具有相互反馈调节功能的p16-Rb-HDAC1-E2F1蛋白信号传导通路。
3．本课题将蛋白质的信号传导与表遗传学结合起来进行研究，可以更全面的阐明遗传信息的精确调控和准确表达方式。我们的研究表明，胆道癌表遗传的改变能够通过对染色质基因转录的调节来影响癌细胞的增殖进程，而表遗传的改变与蛋白信号的传导密切相关。
4．肝外胆管癌和胆囊癌的手术切除率和对放疗、化疗的敏感性均差，本课题以邹声泉教授领导的研究团队的基础研究结果作为理论指导，观察了组蛋白去乙酰化酶抑制剂在体内、体外对胆道癌细胞的抑制作用，为探索新的胆道癌的生物治疗手段进行了有益的尝试。
5．本课题组在邹声泉教授领导下的前期研究表明肝炎病毒感染与肝门胆管癌显著相关并可以引起相应基因的改变。本组资料研究结果表明，p16蛋白在胆囊癌的表达与患者是否合并乙肝具有显著相关性，提示乙型肝炎病毒感染可能参与了胆囊癌变过程中p16基因的缺失或者失活，从而为进一步揭示肝炎病毒在肿瘤癌变中的作用机制提供了新的证据。
Tumorigenesis is a process of multi-factor and multi-step, which involved in the aberration of chromosome structure, inactivation of antioncogene, activation of oncogene, regulation of cell cycle, activation of gene promoter, epigenetics, and so on. Many researches were carried out in these aspects. Regulation of genes became new direction when human genome program had been completed. Regulation and expression of genetic information depend on the effect of protein signals. So study on the protein becomes a hot spot.
Previous studies of Shengquan Zou' group indicate that p16 and HDAC1 play important roles in molecular cytogenetics and epigenetics in extrahepatic cholangiocarcinoma. Studies have demonstrated that Rb/E2F1 pathway influence the activation of HDAC1, and function of Rb/p16 pathway was recognized gradually also. It is a hint that p16, Rb, E2F1 and HDAC1 constitute a protein signal pathway. These protein signals were involved in many aspects of tumor origin and were representative in tumorigenesis. Our research is to study the effect and protein signal pathway of p16-Rb-HDAC1-E2F1 of extrahepatic cholangiocarcinoma and gallbladder carcinoma from many aspects by plasmid transfection, immuneprecipitation, Western blot, RT-PCR, MTT, immunehistochemistry, and so on. To study the correlation between regulation of gene and function of these proteins, which offer the new evidence of the effect of these proteins on biological behaviours of biliary tract cancer. And to analysis the correlation between the expression of these proteins with cinical epidemiology, which offer the new evidence on cause of biliary tract cancer. Although level of our research is limited, we will work further in future. Part I Studies on the interaction among p16-Rb-HDAC1-E2F1 proteins in biliary tract cancer
Thesis 1 The expression of p16-Rb-HDAC1-E2F1 in biliary tract cancer cell lines
Objective To study the expression of proteins and mRNA of p16-Rb-HDAC1-E2F1 in cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line).
Methods Measure the expression of proteins and mRNA of p16-Rb-HDAC1-E2F1 in cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line (Mz-ChA-1 cell line) by immunehistochemistry and RT-PCR assay.
Results
1. There is weak expression of p16 mRNA in QBC_(939) cell line, there is weak expression of Rb mRNA in KMBC cell line, there are weak expressions of HDAC1 mRNA in QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines respectively, there are weak expressions of E2F1 mRNA in QBC_(939), KMBC and Mz-ChA-1 cell lines respectively. There is expression of p16 protein in QBC_(939) cell line, there is expression of Rb protein in KMBC cell line, there are expressions of HDAC1 protein in QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines respectively, there are expressions of E2F1 protein in QBC_(939), KMBC and Mz-ChA-1 cell lines respectively.
2. There is weak expression of p16 protein in tumor tissue of QBC_(939) cell line transplanted nude mice model, there is weak expression of Rb protein in cancer tissue of KMBC cell line transplanted nude mice model. There are expressions of HDAC1 protein in tumor tissue of QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines transplanted nude mice model respectively, but their positive rates are different. There are weak expressions of E2F1 protein in cancer tissue of QBC_(939), KMBC and Mz-ChA-1 cell lines transplanted nude mice model respectively.
Conclusions P16-Rb-HDAC1-E2F1 play some roles in billiary tract
cancer. Biological behaviours of biliary tract cancer cell lines can
be changed by internal environment. Thesis 2 Studies on the interaction among p16-Rb-HDAC1-E2F1
proteins in biliary tract cancer
Objective To study the mechanism and interaction among p16-Rb-HDAC1-E2F1 proteins in biliary tract cancer.
Methods By using the method of immuneprecipitation, we studied the interaction among p16-Rb-HDAC1-E2F1 proteins of extrahepatic cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) which infected with p16 plasmid and pRb plasmid respectively.
Results
1. There are Rb protein(QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines) and E2F1 protein(QBC_(939), KMBC and Mz-ChA-1 cell lines) lie in the precipitation of anti-HDAC1.
2. There are HDAC1 protein(QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines) and E2F1 protein (QBC_(939), KMBC and Mz-ChA-1 cell lines) lie in the precipitation of anti-Rb.
Conclusions
1. Rb protein, HDAC1 protein and E2F1 protein can make the specific interaction in biliary tract cancer, and there are the direct pathways among these protein signals.
2. P16 protein has not direct relationship with Rb protein, HDAC1 protein, and E2F1 protein. Part II Relation between p16-Rb-HDAC1-E2F1 proteins and biological behaviours of biliary tract cancer
Thesis 3 Effect and interaction of p16-Rb-HDAC1-E2F1 proteins in biliary tract cancer cell lines in vivo and in vitro
Objective To study the effect and interaction of p16-Rb-HDAC1-E2F1 on
the growth of cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) in vivo and in vitro.
Methods
1. Transfect p16 plasmid and pRb plasmid into cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line), measure the growth change of biliary tract cancer cell lines after transfection by MTT assay, measure the expression change of pRb mRNA after transfected with p16 plasmid and measure the expression change of p16 mRNA after transfected with pRb plasmid by RT-PCR.
2. By using the same methods to observe the effect of histone deacetylase inhibitor-trichostatin A(TSA),which effect is against HDAC1, on the growth of cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line).
3. Successfully establish the transplanted gallbladder carcinoma nude mice models, and measure the growth change of the transplanted tumor after being transfected and treated with TSA.
Results
1. The expression of Rb mRNA of KMBC cell line transfected with p16 plasmid was inhibited. There are weak expressions of p16 mRNA in OZ and Mz-ChA-1 cell lines transfected with pRb plasmid. 2. The proliferation of cholangiocarcinoam cell lines(KMBC cell line) transfected with p16 plasmid was inhibited, the proliferation of cholangiocarcinoam cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) transfected with pRb plasmid was inhibited, in which the inhibition of proliferation of QBC_(939) cell line transfected with pRb plasmid was the most distinct.
3. TSA can inhibit the proliferation of cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line). These effects were dose-dependent and time-dependent.
4. Successfully established the gallbladder carcinoma transplanted nude mice model, the growth of cancer was inhibited in the nude mice transplanted with gallbladder carcinoma cell line which transfected with pRb plasmid and TSA treatment.
Conclusions
1. There was negative feedback adjustment between p16 and Rb in biliary tract cancer.
2. Rb and TSA can inhibit the growth of gallbladder carcinoma cell line in vivo, Rb and TSA can inhibit the growth of cholangiocarcinoma cell lines and gallbladder carcinoma cell line in vitro.
3. P16 can only inhibit the growth of biliary tract cancer cell lines which express Rb, this indicates that the effect of p16 in biliary tract cancer rely on the function of Rb. Thesis 4 Mechanism of HDAC1 in immune escape of gallbladder carcinoma and correlation between HDAC1 and apoptosis
Objective
1. To study the role of Fas/FasL and HDAC1 in biological behaviours of gallbladder carcinoma.
2. To study their correlated action and mechanism in tumor escape.
Methods
1. Immunohistochemistry technique was used to study the expression of Fas/FasL protein and HDAC1 protein in gallbladder carcinoma tissues, gallbladder adenoma tissues, gallbladder dysplasia tissues and chronic cholecystis tissues.
2. Apoptosis of the infiltrating lymphocytes in these tissues was studied by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method.
3. Expressions of both proteins and apoptosis of the tumor infiltrating lymphocytes in cancer tissues of primary foci were compared with clinicopathological features of gallbladder carcinoma.
4. Study the correlation between Fas/FasL pathway and HDAC1.
Results
1. The positive rates of Fas were not significantly difference among carcinoma, adenoma, dysplasia and chronic cholecystis. The positive rate of FasL in carcinoma was significantly higher than that in chronic cholecystis (X~2=4. 89, P<0.05).
2. The apoptotic index in carcinoma was significantly higher than that in adenoma(t'=4.19, P<0.01)and chronic cholecystis (t' =8. 06,P<0. 01). The apoptotic index was significantly lower in well-differentiated carcinoma and Nevin I -III carcinoma than that in poorly-differentiated carcinoma(t'=2. 63, P<0. 05) and Nevin IV-V carcinoma(t' =3. 33, P<0. 01).
3. The confidence interval of infiltrating lymphocytes in adenoma, chronic cholecystis, well-differentiated carcinoma and Nevin I -III carcinoma was very significantly lower than that in carcinoma(t' =6. 99, P<0. 01), adenoma(t' =3.66, P<0. 01), poorly-differentiated carcinoma (t' =5.31, P<0. 01) and Nevin IV-V carcinoma(t' =3.76, P<0. 01) respectively.
4. The confidence interval of apoptosis of infiltrating lymphocytes in well-differentiated carcinoma was significantly lower than that in poorly-differentiated carcinoma(t=2. 52, P<0. 05), and was not significantly lower in Nevin I-III carcinoma than in Nevin IV-V carcinoma(t=1. 42, P>0.05). Apoptosis of infiltrating lymphocytes was not discovered in adenoma and chronic cholecystis.
5. The expression of FasL and HDAC1 in gallbladder carcinoma displayed some extent positive correlation(P<0. 05), the expression of Fas and HDAC1 in gallbladder carcinoma were not significant correlation.
Conclusions
1. FasL protein expressed in gallbladder carcinoma cells permits tumor cells to escape from immune surveillance of organism by inducing apoptosis in infiltrating lymphocytes of carcinoma tissues. Up-regulation of FasL protein expression plays an important role in invasive depth, histological classification and metastasis of gallbladder carcinoma.
2. The evidence of the role of Fas protein in immune escape of gallbladder carcinoma do not be found.
3. There is the correlation between HDAC1 and immune escape of gallbladder carcinoma. Part III Effect of histone deacetylase inhibitor on p16-Rb-HDAC1-E2F1 proteins signal in biliary tract cancer
Thesis 5 Effect of histone deacetylase inhibitor on
p16-Rb-HDAC1-E2F1 proteins signal in cholangiocarcinoma and gallbladder carcinoma
Objective To study the effect of histone deacetylase inhibitor-trichostatin A(TSA) on p16-Rb-HDAC1-E2F1 signal pathway in extrahepatic cholangiocarcinoma and gallbladder carcinoma.
Methods Treat cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) with TSA, and measure the mRNA expression change of p16, Rb, HDAC1 and E2F1 by RT-PCR assay and the protein expression change by Western blot method. Then transplanted these cells into subcutaneous tissue of nude mice to establish the transplanted cholangiocarcinoma and gallbladder carcinoma nude mice models, and observed the effect of TSA on the expression of Rb、 HDAC1、 DNMT1、 E2F1 of transplanted cancer tissues in vivo.
Results
1. TSA can down-regulate the expression of protein and mRNA of HDAC1 in cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line (Mz-ChA-1 cell line), can up-regulate the expression of protein and mRNA of p16 in KMBC cell line, there are no effect in expression of Rb and E2F1.
2. TSA can not down- or up-regulate the expression of p16, Rb, HDAC1 and E2F1 proteins in the transplanted biliary tract cancer nude mice models.
Conclusions TSA might down-regulate the expression of HDAC1 in cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line), and up-regulate the expression of p16 in Rb~+ cell lines, consequently effect the transcription of gene. The effect of TSA reply on the length of time. Part IV Analysis of the correlation between the p16-Rb-HDAC1-E2F1 proteins and clinicopathological factor in the gallbladder carcinoma
Thesis 6 Analysis of the correlation between the p16-Rb-HDAC1-E2F1 proteins and clinicopathological factor in the gallbladder carcinoma
Objective To study the characteristics of clinicopathology of gallbladder carcinoma and the expression and significance of p16-Rb-HDAC1-E2F1 proteins in gallbladder carcinoma.
Methods
1. Analysis 65 cases of gallbladder carcinoma in Tongji hospital of Huazhong university of science and technology retrospectively (1991-2005).
2. Research the expression and characteristics of p16-Rb-HDAC1-E2F1 proteins respectively existing in tissues of gallbladder carcinoma, gallbladder adenoma, and chronic cholecystitis by immunehistochemistry.
Results
1. The incidence of coexistence of gallstones in gallbladder carcinoma was 44.64%(25/56). The incidence of hepatitis B in gallbladder carcinoma was 26.79%(15/56). The incidence of schistosome in gallbladder carcinoma was 30.36%(17/56). The incidence of multiple pregnancies in gallbladder carcinoma was 72.98% (27/37). Gallbladder carcinoma accounted for 0.64%(56/8807) of cholecystectomy. Preoperative mis-diagnose were often the gallstone, adenoma and liver cancer. Diagnostic rates of B-ultrasound, CT and MRI were 42. 3% (22/52), 57. 1%[ (14-6) / (20-6) ]and 50%[ (5-2) / (8-2) ] respectively. The γ -GT were all positive in gallbladder carcinoma(4/4).
2. The position of the primary carcinoma of gallbladder located in the collum of gallbladder was 29.73%(11/37), in body of gallbladder was 29.73%(11/37), in fundus of gallbladder was 16.22%(6/37). Pathologic histology type of gallbladder carcinoma included adenocarcinoma (83.93%, 47/56), adenoma malignant progression (7.14%, 4/56), squamous cell carcinoma(5.36%, 3/56) and undifferentiated carcinoma (3.57%, 2/56). The incidence of gallbladder carcinoma in Nevin I stage was 14.29% (8/56), IV stage was 14.29%(8/56), V stage was 10.71%(6/56). There were not significant correlation among sex,the primary position and malignant degree of the gallbladder carcinoma. Liver was often infiltrated by tumor(14/56). Nerve bundle of gallbladder wall were infiltrated by two of them.
3. The positive rates of pl6 and Rb proteins in gallbladder carcinoma tissue are respectively higher than that in adenoma tissue and cholecystitis tissue.The positive rates of HDAC1 and E2F1 proteins in gallbladder carcinoma tissue are respectively higher than that in adenoma tissue and cholecystitis tissue. The positive rate of pl6-Rb-HDAC1-E2F1 proteins in gallbladder carcinoma tissue has no relationship with the differentiation degree of gallbladder carcinoma, Nevin stage, age, gender and gallstone. The expression of pl6 in gallbladder carcinoma infected with hepatitis B virus was 27. 7%, and 55. 6% in the others, there was significant different between them(P<0. 05). It took on some extent positive correlation between the expression of pl6 and the expression of Rb in gallbladder carcinoma( γ=0. 6091, t =4. 48, P<0. 001), it took on some extent negative correlation between the expression of pl6 and the expression of HDAC1 in gallbladder carcinoma( γ=-0. 5215, t=3.56, P<0. 005), it took on some extent negative correlation between the expression of Rb and the expression of HDAC1 in gallbladder carcinoma ( γ=-0.3953, t=2.51, P<0. 05).
Conclusions
1. Gallstones, hepatitis B, schistosome and multiple pregnancies are risk factors of gallbladder carcinoma. The γ -GT may be potential tumor marker.
2. Gallbladder carcinoma often locates in collum and body of gallbladder, pathologic histology type of gallbladder carcinoma is often adenocarcinoma, tumor can infiltrate along nerve bundle of gallbladder wall. There are not significant correlation among sex, the primary position and malignant degree of the gallbladder carcinoma.
3. The expression of p16-Rb-HDAC1-E2F1 proteins in gallbladder carcinoma tissue has no relationship with the differentiation degree of gallbladder carcinoma, Nevin stage, age, and gender.
4. The expression of p16 protein in gallbladder carcinoma tissue is significantly correlative with hepatitis B.
5. The down-regulating expression of p16 protein and Rb protein and down-regulating expression of HDAC1 protein and E2F1 protein is relative with the gallbladder carcinoma pathological characteristics, there are interaction among p16 protein, Rb protein and HDAC1 protein of the gallbladder carcinoma. Thesis 7 Analysis of the correlation between the p16-Rb-HDAC1-E2F1 proteins and clinicopathological factor in the unsuspected gallbladder carcinoma
Objective To study the characteristics of clinicopathology in unsuspected gallbladder carcinoma and the expression and significance of p16-Rb-HDAC1-E2F1 proteins in unsuspected gallbladder carcinoma.
Methods Analysis 23 cases of unsuspected gallbladder carcinoma in Tongji hospital retrospectively, characteristics of clinicopathology were compared with preoperative diagnosed gallbladder carcinoma in same period.Research the expression and characteristics of p16-Rb-HDAC1-E2F1 proteins respectively existing in tissues of unsuspected gallbladder carcinoma by immunehistochemistry.
Results The incidence of coexistence of gallstones in unsuspected gallbladder carcinoma was significantly higher than in preoperative diagnosed gallbladder carcinoma(P<0. 01). The incidence of hepatitis B in unsuspected gallbladder carcinoma was 21. 74%(5/23), in preoperative diagnosed gallbladder carcinoma was 30. 30%(10/33). The incidence of schistosome in unsuspected gallbladder carcinoma was 39. 13%(9/23), in preoperative diagnosed gallbladder carcinoma was 24.24%(8/33). The incidence of multiple pregnancies in unsuspected gallbladder carcinoma was 56. 52%(13/23), in preoperative diagnosed gallbladder carcinoma was 42. 42%(14/33). The γ-GT were all positive in unsuspected gallbladder carcinoma(2/2) and preoperative diagnosed gallbladder carcinoma(2/2). The position of the primary carcinoma of unsuspected gallbladder carcinoma ofen located in the collum and body of the gallbladder, the position of the primary carcinoma of preoperative diagnosed gallbladder carcinoma ofen located in the body and fundus of the gallbladder. The incidence of Nevin I stage and II stage in unsuspected gallbladder carcinoma was significantly higher than that in preoperative diagnosed gallbladder carcinoma(P<0. 05). The incidence of Nevin IV stage in unsuspected gallbladder carcinoma was significantly lower than that in preoperative diagnosed gallbladder carcinoma(P<0. 05). The incidence of Nevin V stage in unsuspected gallbladder carcinoma was significantly lower than that in preoperative diagnosed gallbladder carcinoma(P<0. 01). There was not significant difference between expression of p16-Rb-HDAC1-E2F1 proteins of unsuspected gallbladder carcinoma and expression of p16-Rb-HDAC1-E2F1 proteins of preoperative diagnosed gallbladder carcinoma.
Conclusions Unsuspected gallbladder carcinoma is different from preoperative diagnosed gallbladder carcinoma in the position of the primary carcinoma, the malignant degree and the incidence of coexistence of gallstones. Gallstones, hepatitis B , schistosome and multiple pregnancies are risk factors of unsuspected gallbladder carcinoma. Unsuspected gallbladder carcinoma has similar molecular biological characteristics with preoperative diagnosed gallbladder carcinoma. Ingenuity in this study:
1. The protein signals of p16-Rb-HDAC1-E2F1 in biliary tract cancer involved in the inactivation of antioncogene, regulation of cell cycle, histone acetylation activation of gene promoter, epigenetics, and so on, which was representative in tumorigenesis. The research methods we used have combined these aspects, which apply a available pattern in studying the pathogenesis of cancer.
2. Our study displayed that Rb protein, HDAC1 protein and E2F1 protein can make the specific interaction in biliary tract cancer, and there are the indirect pathways among p16 protein signals and the others, the effect of pl6 in biliary tract cancer rely on the function of Rb, which indicates that p16, Rb, E2F1 and HDAC1 constitute a protein signal pathway.
3. Our study connects the protein signal conduction with the epigenetics, which may clarify the regulation of gene even more. Our study display that the epigenetics can influence cell differentiation by regulation of gene, and the change of the epigenetics connects with protein signals.
4. To extrahepatic cholangiocarcinoma and gallbladder carcinoma, the rate of excision is low and the sensitivity to chemotherapy and actinotherapy is poor. We study the effect of histone deacetylase inhibitor on the growth of biliary tract cancer in vivo and in vitro based on the previous datas of Shengquan Zou' group, which was valuable experiment in biological treatment of biliary tract cancer.
5. The previous datas of Shengquan Zou' group indicate that hepatitis
virus may influence the expression of gene in cholangiocarcinoma. Our research indicates that the expression of pl6 protein in gallbladder carcinoma is significantly correlative with hepatitis B.
在邹声泉教授的领导下，我们的课题组在前期的研究中发现抑癌基因p16和组蛋白乙酰化酶HDAC1与肝外胆管癌的细胞分子遗传学和表遗传学有关联，提示其在肝外胆管癌的发生和发展中扮演着重要角色。另有研究认为，Rb／E2F1途径是细胞周期调控的中心环节并可以调节HDAC1的活性进而影响DNA乙酰化，Rh／p16途径在肿瘤中的作用也逐渐得到公认。这些研究结果提示，p16、Rb、HDAC1和E2F1之间可能在胆道肿瘤中存在一个具有相互反馈调节功能的蛋白信号传导通路。这些蛋白信号涉及抑癌基因的失活、组蛋白乙酰化、基因启动子转录活化和肿瘤表遗传学等诸多方面，所以其在肿瘤演变过程中极具有代表性。
为此本课题从研究肝外胆管癌和胆囊癌p16、Rh、HDAC1和E2F1之间的蛋白信号传导着手，应用质粒转染、免疫沉淀、Western blot、RT-PCR、MTT和免疫组化等实验方法，从细胞水平到组织水平，从体外作用到体内作用，多角度、多层面的研究这些蛋白信号的传导途径及其蛋白质之间的相互作用，探讨这些蛋白的功能及其与肿瘤的基因调控和表遗传学等方面的关系，期望能够为阐明其在胆道癌中的作用机制提供新的理论基础和实验依据。同时将这些蛋白质的表达特征与胆道癌的临床流行病学相联系，期望能从众多纷繁的临床特征中找到与这些蛋白质相关联的因素，从而为寻找胆道癌的病因提供新的证据。但由于时间的关系，本课题尚局限于较浅的层面，在今后的工作中我们将进一步深入研究。
第一部分胆道癌p16-Rb-HDAC1-E2F1中各种蛋白质之间的相互作用及其信号传导途径的研究
论文1 p16-Rb-HDAC1-E2F1在胆道癌细胞系中的表达
目的研究p16-Rb-HDAC1-E2F1中各种蛋白及其mRNA在肝外胆管癌和胆囊癌细胞系中的表达情况。
方法用免疫组织化学和RT-PCR方法检测肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)中p16-Rb-HDAC1-E2F1蛋白及其mRNA的表达。
结果
1．P16 mRNA在QBC_(939)细胞有弱表达，在KMBC细胞、Mz-ChA-1细胞和OZ细胞无表达；Rb mRNA在KMBC细胞有弱表达，在QBC_(939)细胞、Mz-ChA-1细胞和OZ细胞无表达；HDAC1 mRNA在QBC_(939)细胞、KMBC细胞、OZ细胞和Mz-ChA-1细胞均有弱表达；E2F1 mRNA在QBC_(939)细胞、KMBC细胞和Mz-ChA-1细胞有弱表达，在OZ细胞无表达。p16蛋白在QBC_(939)细胞有表达；Rb蛋白在KMBC细胞有表达；HDAC1蛋白在QBC_(939)细胞、KMBC细胞、OZ细胞和Mz-ChA-1细胞均有表达；E2F1蛋白在QBC_(939)细胞、KMBC细胞和Mz-ChA-1细胞有表达。
2．P16蛋白在QBC_(939)细胞种植瘤中有弱表达；Rb蛋白在KMBC细胞种植瘤中有弱表达；HDAC1蛋白在QBC_(939)、KMBC、OZ和Mz-ChA-1细胞种植瘤中有表达，但表达丰度在同种细胞种植瘤中不同；E2F1蛋白在QBC_(939)、KMBC和Mz-ChA-1细胞种植瘤中有弱表达。
结论P16、Rb、HDAC1、E2F1参与了胆道癌的进程；体内环境可对胆道癌细胞系的生物学特性产生影响。
论文2胆道癌p16-Rb-HDAC1-E2F1中各种蛋白质之间的相互作用研究
目的研究胆道癌p16-Rb-HDAC1-E2F1中各种蛋白质之间的相互作用及其机理。
方法用免疫沉淀法检测分别转染p16质粒和pRb质粒的肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)的p16-Rb-HDAC1-E2F1中各种蛋白质之间的相互作用。
结果
1．用HDAC1抗体免疫沉淀下来的蛋白中有Rb蛋白(在QBC_(939)、KMBC、OZ和Mz-ChA-1细胞系)和E2F1蛋白(在QBC_(939)、KMBC和Mz-ChA-1细胞系)的存在，无p16蛋白存在。
2．用Rh抗体免疫沉淀下来的蛋白中有HDAC1蛋白(在QBC_(939)、KMBC、OZ和Mz-ChA-1细胞系)和E2F1蛋白(在QBC_(939)、KMBC和Mz-ChA-1细胞系)的存在，无p16蛋白存在。
结论
1．胆道癌Rb蛋白、HDAC1蛋白和E2F1蛋白之间在细胞水平存在特异的相互作用，表明其蛋白信号之间存在直接的传导途径。
2．P16蛋白在胆道癌中与Rb蛋白、HDAC1蛋白和E2F1蛋白之间并不存在直接的相互作用。
第二部分p16-Rb-HDAC1-E2F1蛋白与胆道癌生物学特性的关系
论文3 p16-Rb-HDAC1-E2F1蛋白在胆道癌细胞中的作用及其相互影响
目的研究p16-Rb-HDAC1-E2F1在体内、体外对肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)生长的作用及其各蛋白之间的相互影响。
方法
1．转染p16质粒和pRh质粒进入肝外胆管癌细胞系(QBC_(939)、KMBC、OZ celllines)和胆囊癌细胞系(Mz-ChA-1 cell line)，用MTT法检测转染前后各胆道癌细胞系生长的变化，用RT-PCR法检测转染p16质粒前后各胆道癌细胞系Rb mRNA的表达变化和转染pRb质粒前后各胆道癌细胞系p16 mRNA的表达变化。
2．用与HDAC1功能相反的去乙酰化酶抑制剂-曲古抑菌素(trichostatinA，TSA)作用于肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)，用MTT法检测TSA对这些胆道癌细胞系生长的影响。
3．分别将转染p16质粒、pRb质粒和经TSA处理的胆囊癌细胞系(Mz-ChA-1cell line)接种裸鼠皮下建立胆囊癌裸鼠种植模型，观察其转染p16质粒和pRb质粒前后和TSA处理前后裸鼠体内种植瘤生长的变化。
结果
1．转染p16质粒的KMBC细胞的Rb mRNA的表达受到抑制。转染pRb质粒的OZ和Mz-ChA-1细胞的p16 mRNA出现弱表达，QBC_(939)细胞和KMBC细胞的p16mRNA的表达无变化。
论文4 HDAC1与凋亡在胆囊癌中的相关性及其在免疫逃逸机制中的作用研究
目的
1．研究凋亡的死亡信号受体途径(Fas／FasL途径)和组蛋白去乙酰化酶HDAC1在胆囊癌生物学行为中的作用。
2．探讨HDAC1在胆囊癌免疫逃逸机制中的相关作用和机制。
方法
1．应用免疫组织化学方法研究胆囊癌组织、胆囊腺瘤组织、胆囊上皮不典型增生组织和慢性胆囊炎组织中Fas／FasL蛋白的表达和HDAC1蛋白的表达情况。
2．运用TUNEL法检测在胆囊癌组织、胆囊腺瘤组织、胆囊上皮不典型增生组织和慢性胆囊炎组织中的原位组织细胞的凋亡情况及其浸润的淋巴细胞的凋亡情况。
3．比较在不同的胆囊癌临床病理类型中Fas蛋白和FasL蛋白的表达情况的差异和在原位胆囊癌组织浸润的淋巴细胞的凋亡情况的差异。
4．探讨胆囊癌Fas／FasL蛋白的表达与组蛋白去乙酰化酶HDAC1蛋白的表达的相关性。
结果
1．Fas蛋白表达的阳性率在胆囊癌组织、胆囊腺瘤组织、胆囊上皮不典型增生组织和慢性胆囊炎组织之间的差异无显著意义。FasL蛋白表达的阳性率在胆囊癌组织显著高于慢性胆囊炎组织(X~2=4.89，P＜0.05)。
2．胆囊癌的组织细胞的凋亡指数显著高于胆囊腺瘤的组织细胞(t′=4.19，P＜0.01)和慢性胆囊炎的组织细胞(t′=8.06，P＜0.01)的凋亡指数；高分化的胆囊癌的组织细胞的凋亡指数显著低于低分化的胆囊癌的组织细胞的凋亡指数(t′=2.63，P＜0.05)，NevinⅠ、Ⅱ、Ⅲ期的胆囊癌的组织细胞的凋亡指数显著低于NevinⅣ、Ⅴ期的胆囊癌的组织细胞的凋亡指数(t′=3.33，P＜0.01)。
3．浸润的淋巴细胞的数量在胆囊腺瘤组织显著低于在胆囊癌组织(t′=6.99，P＜0.01)，在慢性胆囊炎组织显著低于在胆囊腺瘤组织(t′=3.66，P＜0.01)；在高分化胆囊癌组织显著低于在低分化胆囊癌组织(t′=5.31，P＜0.01)，在胆囊癌NevinⅠ、Ⅱ、Ⅲ期组织显著低于在胆囊癌NevinⅣ、Ⅴ期组织(t=-3.76，P＜0.01)。
4．浸润的淋巴细胞的凋亡数量在高分化的胆囊癌组织显著低于在低分化的胆囊癌组织(t=-2.52，P＜0.05)；在NevinⅠ、Ⅱ、Ⅲ期的胆囊癌组织中浸润的淋巴细胞的凋亡数量与在NevinⅣ、Ⅴ期的胆囊癌组织浸润的淋巴细胞的凋亡数量之间的差异无显著意义(t=-1.42，P＞0.05)；在胆囊腺瘤组织中和慢性胆囊炎组织中未见到浸润的淋巴细胞的凋亡。
5．HDAC1蛋白和FasL蛋白在胆囊癌组织的表达呈显著正相关(P＜0.05)，HDAC1蛋白和Fas蛋白在胆囊癌组织的表达无显著相关性。
结论
1．胆囊癌组织细胞表达的FasL通过诱导在癌组织浸润的淋巴细胞的凋亡从而使癌细胞逃避机体的免疫监视，它的上调表达在胆囊癌的分化、浸润和转移过程中扮演着重要角色。
2．未证实胆囊癌组织细胞表达的Fas蛋白在胆囊癌的免疫逃逸中具有作用。
3．组蛋白去乙酰化酶HDAC1蛋白和参与胆囊癌免疫逃逸的FasL蛋白在胆囊癌组织的表达呈显著正相关，提示我们HDAC1与胆囊癌的免疫逃逸具有一定的相关性。
第三部分组蛋白去乙酰化酶抑制剂对胆道癌p16-Rb-HDAC1-E2F1蛋白信号传导的影响
论文5组蛋白去乙酰化酶抑制剂对肝外胆管癌细胞和胆囊癌细胞p16-Rb-HDAC1-E2F1蛋白信号传导的影响
目的研究组蛋白去乙酰化酶抑制剂-曲古抑菌素(trichostatin A，TSA)对肝外胆管癌和胆囊癌p16-Rh-HDAC1-E2F1蛋白信号传导的影响。
方法用TSA作用于肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lines)和胆囊癌细胞系(Mz-ChA-1 cell line)，然后用RT-PCR检测p16、Rb、HDAC1、E2F1之mRNA的表达变化，用Western blot检测其蛋白的表达变化。将这些细胞接种在裸鼠皮下建立肝外胆管癌和胆囊癌裸鼠种植瘤模型，用免疫组织化学方法观察TSA在体内对裸鼠种植瘤组织中Rb、HDAC1、DNMT1、E2F1蛋白表达的影响。
结果
1．TSA可以减弱肝外胆管癌细胞系(QBC_(939)、KMBC、OZ cell lities)和胆囊癌细胞系(Mz-ChA-1 cell line)HDAC1 mRNA和蛋白的表达；增强KMBC细胞系p16 mRNA和蛋白的表达；对各细胞系Rh和E2F1 mRNA和蛋白的表达无影响。
2．TSA作用前后的肝外胆管癌细胞系和胆囊癌细胞系裸鼠成瘤组织中的各种蛋白的表达均无变化。
结论TSA可以抑制肝外胆管癌和胆囊癌HDAC1的表达，并在Rb阳性的细胞中促进p16的表达，从而进一步影响遗传信息的转录。TSA的作用存在时效性。
第四部分p16-Rb-HDAC1-E2F1蛋白与胆囊癌临床病理学的关系
论文6 p16-Rb-HDAC1-E2F1蛋白与胆囊癌临床病理学因素的关系分析
目的探讨胆囊癌的临床病理学特征并研究p16-Rb-HDAC1-E2F1蛋白与胆囊癌临床病理学因素的关系及其意义。
方法
1．回顾性分析华中科技大学附属同济医院1991-2005年收治的65例胆囊癌病例。
2．应用免疫组化法研究胆囊痛组织中p16-Rb-HDAC1-E2F1蛋白的表达水平及特征。
结果
1．本组胆囊癌合并胆囊结石的比例为44.64％(25／56)。胆囊癌患者有乙肝病史的比例为26.79％(15／56)，有血吸虫疫水接触史的比例为30.36％(17／56)。多产者比例为72.98％(27／37)。本组胆囊癌病例占同期胆囊切除术总数的0.64％(56／8807)。术前误诊疾病依次为结石、腺瘤和肝癌。影像学检查中，B超的正确率为42.3％(22／52)，CT的正确率为57.1％[(14-6)／(20-6)]，MRI的正确率为50％[(5-2)／(8-2)]。有4例胆囊癌患者检查了血γ-GT，其结果均高于正常值。
2．本组胆囊癌原发部位多位于胆囊颈部(29.73％，11／37)和体部(29.73％，11／37)，其次为胆囊底部(16.22％，6／37)，位于胆囊管部占5.41％(2／37)，位于胆囊体部和底部占13.51％(5／37)，另有5.41％(2／37)位于胆囊颈体交界部。胆囊癌组织学分型依次为腺瘤(83.93％，47／56)、腺瘤恶变(7.14％，4／56)、鳞癌(5.36％，3／56)和未分化癌(3.57％，2／56)。本组胆囊癌Nevin分期，Ⅰ期占14.29％(8／56)，Ⅱ期占10.71％(6／56)，Ⅲ期占26.79％(15／56)，Ⅳ期占14.29％(8／56)。胆囊癌分级，高分化占44.64％(25／56)，中分化占12.5％(7／56)，低分化占42.86％(24／56)。本组病例的胆囊癌的原发部位与肿瘤的分级、分期和患者性别均无显著相关性。胆囊癌转移部位多为肝脏(14／56)。有2例肿瘤组织浸润胆囊壁内神经束衣。
3．P16蛋白和Rb蛋白在胆囊癌组织中低表达，HDAC1蛋白和E2F1蛋白在胆囊癌组织中高表达，其表达均与胆囊癌的分化程度、Nevin分期、患者的年龄、性别和是否合并结石无显著相关性。P16蛋白在胆囊癌的表达率在合并乙肝者为27.7％，在没有合并乙肝者为55.6％，二者之间的差异具有显著性意义(P＜0.05)；Rb蛋白、HDAC1蛋白和E2F1蛋白在胆囊癌的表达均与患者是否合并乙肝无显著相关性。P16蛋白和Rb蛋白在胆囊癌的表达呈显著正相关(γ=0.6091，t=4.48，P＜0.001)，p16蛋白和HDAC1蛋白在胆囊癌的表达呈显著负相关(γ=-0.5215，t=3.56，P＜0.005)，Rb蛋白和HDAC1蛋白在胆囊癌的表达呈显著负相关(γ=-0.3953，t=2.51，P＜0.05)，E2F1蛋白和其它蛋白在胆囊癌的表达无显著相关性。
结论
1．胆囊结石、乙肝病毒感染、血吸虫感染和多产妇是胆囊癌的高危因素；γ-GT可能是胆囊癌有潜力的肿瘤标志物。
2．胆囊癌多位于胆囊颈部和体部，组织分型以腺癌居多，并具有沿神经浸润的特性；胆囊癌的恶性程度与其原发部位和患者性别无关。
3．P16蛋白、Rb蛋白、HDAC1蛋白和E2F1蛋白在胆囊癌的表达均与肿瘤的恶性程度、患者的年龄和性别无显著相关性。
4．P16蛋白在胆囊癌的表达与患者是否合并乙肝具有显著相关性。
5．P16蛋白、Rb蛋白的低表达和HDAC1蛋白、E2F1蛋白的高表达参与了胆囊癌的进程，并且p16蛋白、Rb蛋白和HDAC1蛋白在胆囊癌中具有相互协同作用。
目的探讨意外胆囊癌的临床病理学特征并研究p16-Rb-HDAC1-E2F1蛋白与意外胆囊癌临床病理学因素的关系及其意义。
方法回顾性研究同济医院23例意外胆囊癌病例，并和同期收治的33例术前确诊的胆囊癌病例进行比较分析，并应用免疫组化法研究意外胆囊癌组织中p16-Rb-HDAC1-E2F1蛋白的表达水平及特征。
结果意外胆囊癌合并胆囊结石的比例高于术前确诊的胆囊癌，差异有非常显著意义(P＜0.01)。患者有乙肝病史的比例在意外胆囊癌为21.74％(5／23)，在术前确诊的胆囊癌为30.30％(10／33)。患者有血吸虫疫水接触史的比例在意外胆囊癌为39.13％(9／23)，在术前确诊的胆囊癌为24.24％(8／33)。多产(大于三胎)的比例在意外胆囊癌为56.52％(13／23)，在术前确诊的胆囊癌为42.42％(14／33)。分别有2例意外胆囊癌和2例术前确诊的胆囊癌检查了血．γ-GT，其结果均高于正常值。意外胆囊癌的原发部位多位于胆囊颈部和体部，术前确诊的胆囊癌原发部位多位于胆囊体部和底部。意外胆囊癌的Nevin分期，在Ⅰ期和Ⅱ期的比例均高于术前确诊的胆囊癌，差异有显著意义(P＜0.05)；在Ⅳ期的比例低于术前确诊的胆囊癌，差异有显著意义(P＜0.05)；在Ⅴ期的比例低于术前确诊的胆囊癌，差异有非常显著意义(P＜0.01)。意外胆囊癌的分级，高分化者高于术前确诊的胆囊癌，差异有显著意义(P＜0.05)；低分化者低于术前确诊的胆囊癌，差异有显著意义(P＜0.05)。p16-Rb-HDAC1-E2F1蛋白的表达在意外胆囊癌和非意外胆囊癌的差异无显著性意义。
结论意外胆囊癌具有肿瘤原发部位以胆囊颈、体部居多、恶性程度较术前确诊的胆囊癌低和结石的并存率较术前确诊的胆囊癌高的特征；结石、乙肝、血吸虫和多产是意外胆囊癌的高危因素。意外胆囊癌和非意外胆囊癌具有相似的分子生物学特征。
本课题从研究蛋白质的功能着手，探讨了肝外胆管癌和胆囊癌p16-Rb-HDAC1-E2F1之间的蛋白信号传导途径及其蛋白质之间的相互作用，得出以下结论和创新点：
1．胆道癌p16-Rb-HDAC1-E2F1之间的蛋白信号传导涉及抑癌基因的失活、细胞周期的调控、组蛋白乙酰化、基因启动子的转录活化和肿瘤表遗传学等几个方面，本课题将它们结合起来进行研究，更有代表性的说明胆道癌演变过程的复杂性。
2．胆道癌Rb蛋白、HDAC1蛋白和E2F1蛋白之间在细胞水平存在特异的相互作用，表明其蛋白信号之间存在直接的传导途径。p16蛋白在胆道癌中与Rb蛋白、HDAC1蛋白和E2F1蛋白之间存在间接的相互作用，并且证实p16对胆道癌的抑制作用依赖于Rb的功能。提示在胆道癌中存在一个具有相互反馈调节功能的p16-Rb-HDAC1-E2F1蛋白信号传导通路。
3．本课题将蛋白质的信号传导与表遗传学结合起来进行研究，可以更全面的阐明遗传信息的精确调控和准确表达方式。我们的研究表明，胆道癌表遗传的改变能够通过对染色质基因转录的调节来影响癌细胞的增殖进程，而表遗传的改变与蛋白信号的传导密切相关。
4．肝外胆管癌和胆囊癌的手术切除率和对放疗、化疗的敏感性均差，本课题以邹声泉教授领导的研究团队的基础研究结果作为理论指导，观察了组蛋白去乙酰化酶抑制剂在体内、体外对胆道癌细胞的抑制作用，为探索新的胆道癌的生物治疗手段进行了有益的尝试。
5．本课题组在邹声泉教授领导下的前期研究表明肝炎病毒感染与肝门胆管癌显著相关并可以引起相应基因的改变。本组资料研究结果表明，p16蛋白在胆囊癌的表达与患者是否合并乙肝具有显著相关性，提示乙型肝炎病毒感染可能参与了胆囊癌变过程中p16基因的缺失或者失活，从而为进一步揭示肝炎病毒在肿瘤癌变中的作用机制提供了新的证据。
Tumorigenesis is a process of multi-factor and multi-step, which involved in the aberration of chromosome structure, inactivation of antioncogene, activation of oncogene, regulation of cell cycle, activation of gene promoter, epigenetics, and so on. Many researches were carried out in these aspects. Regulation of genes became new direction when human genome program had been completed. Regulation and expression of genetic information depend on the effect of protein signals. So study on the protein becomes a hot spot.
Previous studies of Shengquan Zou' group indicate that p16 and HDAC1 play important roles in molecular cytogenetics and epigenetics in extrahepatic cholangiocarcinoma. Studies have demonstrated that Rb/E2F1 pathway influence the activation of HDAC1, and function of Rb/p16 pathway was recognized gradually also. It is a hint that p16, Rb, E2F1 and HDAC1 constitute a protein signal pathway. These protein signals were involved in many aspects of tumor origin and were representative in tumorigenesis. Our research is to study the effect and protein signal pathway of p16-Rb-HDAC1-E2F1 of extrahepatic cholangiocarcinoma and gallbladder carcinoma from many aspects by plasmid transfection, immuneprecipitation, Western blot, RT-PCR, MTT, immunehistochemistry, and so on. To study the correlation between regulation of gene and function of these proteins, which offer the new evidence of the effect of these proteins on biological behaviours of biliary tract cancer. And to analysis the correlation between the expression of these proteins with cinical epidemiology, which offer the new evidence on cause of biliary tract cancer. Although level of our research is limited, we will work further in future. Part I Studies on the interaction among p16-Rb-HDAC1-E2F1 proteins in biliary tract cancer
Thesis 1 The expression of p16-Rb-HDAC1-E2F1 in biliary tract cancer cell lines
Objective To study the expression of proteins and mRNA of p16-Rb-HDAC1-E2F1 in cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line).
Methods Measure the expression of proteins and mRNA of p16-Rb-HDAC1-E2F1 in cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line (Mz-ChA-1 cell line) by immunehistochemistry and RT-PCR assay.
Results
1. There is weak expression of p16 mRNA in QBC_(939) cell line, there is weak expression of Rb mRNA in KMBC cell line, there are weak expressions of HDAC1 mRNA in QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines respectively, there are weak expressions of E2F1 mRNA in QBC_(939), KMBC and Mz-ChA-1 cell lines respectively. There is expression of p16 protein in QBC_(939) cell line, there is expression of Rb protein in KMBC cell line, there are expressions of HDAC1 protein in QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines respectively, there are expressions of E2F1 protein in QBC_(939), KMBC and Mz-ChA-1 cell lines respectively.
2. There is weak expression of p16 protein in tumor tissue of QBC_(939) cell line transplanted nude mice model, there is weak expression of Rb protein in cancer tissue of KMBC cell line transplanted nude mice model. There are expressions of HDAC1 protein in tumor tissue of QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines transplanted nude mice model respectively, but their positive rates are different. There are weak expressions of E2F1 protein in cancer tissue of QBC_(939), KMBC and Mz-ChA-1 cell lines transplanted nude mice model respectively.
Conclusions P16-Rb-HDAC1-E2F1 play some roles in billiary tract
cancer. Biological behaviours of biliary tract cancer cell lines can
be changed by internal environment. Thesis 2 Studies on the interaction among p16-Rb-HDAC1-E2F1
proteins in biliary tract cancer
Objective To study the mechanism and interaction among p16-Rb-HDAC1-E2F1 proteins in biliary tract cancer.
Methods By using the method of immuneprecipitation, we studied the interaction among p16-Rb-HDAC1-E2F1 proteins of extrahepatic cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) which infected with p16 plasmid and pRb plasmid respectively.
Results
1. There are Rb protein(QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines) and E2F1 protein(QBC_(939), KMBC and Mz-ChA-1 cell lines) lie in the precipitation of anti-HDAC1.
2. There are HDAC1 protein(QBC_(939), KMBC, OZ and Mz-ChA-1 cell lines) and E2F1 protein (QBC_(939), KMBC and Mz-ChA-1 cell lines) lie in the precipitation of anti-Rb.
Conclusions
1. Rb protein, HDAC1 protein and E2F1 protein can make the specific interaction in biliary tract cancer, and there are the direct pathways among these protein signals.
2. P16 protein has not direct relationship with Rb protein, HDAC1 protein, and E2F1 protein. Part II Relation between p16-Rb-HDAC1-E2F1 proteins and biological behaviours of biliary tract cancer
Thesis 3 Effect and interaction of p16-Rb-HDAC1-E2F1 proteins in biliary tract cancer cell lines in vivo and in vitro
Objective To study the effect and interaction of p16-Rb-HDAC1-E2F1 on
the growth of cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) in vivo and in vitro.
Methods
1. Transfect p16 plasmid and pRb plasmid into cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line), measure the growth change of biliary tract cancer cell lines after transfection by MTT assay, measure the expression change of pRb mRNA after transfected with p16 plasmid and measure the expression change of p16 mRNA after transfected with pRb plasmid by RT-PCR.
2. By using the same methods to observe the effect of histone deacetylase inhibitor-trichostatin A(TSA),which effect is against HDAC1, on the growth of cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line).
3. Successfully establish the transplanted gallbladder carcinoma nude mice models, and measure the growth change of the transplanted tumor after being transfected and treated with TSA.
Results
1. The expression of Rb mRNA of KMBC cell line transfected with p16 plasmid was inhibited. There are weak expressions of p16 mRNA in OZ and Mz-ChA-1 cell lines transfected with pRb plasmid. 2. The proliferation of cholangiocarcinoam cell lines(KMBC cell line) transfected with p16 plasmid was inhibited, the proliferation of cholangiocarcinoam cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) transfected with pRb plasmid was inhibited, in which the inhibition of proliferation of QBC_(939) cell line transfected with pRb plasmid was the most distinct.
3. TSA can inhibit the proliferation of cholangiocarcinoma cell lines (QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line). These effects were dose-dependent and time-dependent.
4. Successfully established the gallbladder carcinoma transplanted nude mice model, the growth of cancer was inhibited in the nude mice transplanted with gallbladder carcinoma cell line which transfected with pRb plasmid and TSA treatment.
Conclusions
1. There was negative feedback adjustment between p16 and Rb in biliary tract cancer.
2. Rb and TSA can inhibit the growth of gallbladder carcinoma cell line in vivo, Rb and TSA can inhibit the growth of cholangiocarcinoma cell lines and gallbladder carcinoma cell line in vitro.
3. P16 can only inhibit the growth of biliary tract cancer cell lines which express Rb, this indicates that the effect of p16 in biliary tract cancer rely on the function of Rb. Thesis 4 Mechanism of HDAC1 in immune escape of gallbladder carcinoma and correlation between HDAC1 and apoptosis
Objective
1. To study the role of Fas/FasL and HDAC1 in biological behaviours of gallbladder carcinoma.
2. To study their correlated action and mechanism in tumor escape.
Methods
1. Immunohistochemistry technique was used to study the expression of Fas/FasL protein and HDAC1 protein in gallbladder carcinoma tissues, gallbladder adenoma tissues, gallbladder dysplasia tissues and chronic cholecystis tissues.
2. Apoptosis of the infiltrating lymphocytes in these tissues was studied by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method.
3. Expressions of both proteins and apoptosis of the tumor infiltrating lymphocytes in cancer tissues of primary foci were compared with clinicopathological features of gallbladder carcinoma.
4. Study the correlation between Fas/FasL pathway and HDAC1.
Results
1. The positive rates of Fas were not significantly difference among carcinoma, adenoma, dysplasia and chronic cholecystis. The positive rate of FasL in carcinoma was significantly higher than that in chronic cholecystis (X~2=4. 89, P<0.05).
2. The apoptotic index in carcinoma was significantly higher than that in adenoma(t'=4.19, P<0.01)and chronic cholecystis (t' =8. 06,P<0. 01). The apoptotic index was significantly lower in well-differentiated carcinoma and Nevin I -III carcinoma than that in poorly-differentiated carcinoma(t'=2. 63, P<0. 05) and Nevin IV-V carcinoma(t' =3. 33, P<0. 01).
3. The confidence interval of infiltrating lymphocytes in adenoma, chronic cholecystis, well-differentiated carcinoma and Nevin I -III carcinoma was very significantly lower than that in carcinoma(t' =6. 99, P<0. 01), adenoma(t' =3.66, P<0. 01), poorly-differentiated carcinoma (t' =5.31, P<0. 01) and Nevin IV-V carcinoma(t' =3.76, P<0. 01) respectively.
4. The confidence interval of apoptosis of infiltrating lymphocytes in well-differentiated carcinoma was significantly lower than that in poorly-differentiated carcinoma(t=2. 52, P<0. 05), and was not significantly lower in Nevin I-III carcinoma than in Nevin IV-V carcinoma(t=1. 42, P>0.05). Apoptosis of infiltrating lymphocytes was not discovered in adenoma and chronic cholecystis.
5. The expression of FasL and HDAC1 in gallbladder carcinoma displayed some extent positive correlation(P<0. 05), the expression of Fas and HDAC1 in gallbladder carcinoma were not significant correlation.
Conclusions
1. FasL protein expressed in gallbladder carcinoma cells permits tumor cells to escape from immune surveillance of organism by inducing apoptosis in infiltrating lymphocytes of carcinoma tissues. Up-regulation of FasL protein expression plays an important role in invasive depth, histological classification and metastasis of gallbladder carcinoma.
2. The evidence of the role of Fas protein in immune escape of gallbladder carcinoma do not be found.
3. There is the correlation between HDAC1 and immune escape of gallbladder carcinoma. Part III Effect of histone deacetylase inhibitor on p16-Rb-HDAC1-E2F1 proteins signal in biliary tract cancer
Thesis 5 Effect of histone deacetylase inhibitor on
p16-Rb-HDAC1-E2F1 proteins signal in cholangiocarcinoma and gallbladder carcinoma
Objective To study the effect of histone deacetylase inhibitor-trichostatin A(TSA) on p16-Rb-HDAC1-E2F1 signal pathway in extrahepatic cholangiocarcinoma and gallbladder carcinoma.
Methods Treat cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) with TSA, and measure the mRNA expression change of p16, Rb, HDAC1 and E2F1 by RT-PCR assay and the protein expression change by Western blot method. Then transplanted these cells into subcutaneous tissue of nude mice to establish the transplanted cholangiocarcinoma and gallbladder carcinoma nude mice models, and observed the effect of TSA on the expression of Rb、 HDAC1、 DNMT1、 E2F1 of transplanted cancer tissues in vivo.
Results
1. TSA can down-regulate the expression of protein and mRNA of HDAC1 in cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line (Mz-ChA-1 cell line), can up-regulate the expression of protein and mRNA of p16 in KMBC cell line, there are no effect in expression of Rb and E2F1.
2. TSA can not down- or up-regulate the expression of p16, Rb, HDAC1 and E2F1 proteins in the transplanted biliary tract cancer nude mice models.
Conclusions TSA might down-regulate the expression of HDAC1 in cholangiocarcinoma cell lines(QBC_(939), KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line), and up-regulate the expression of p16 in Rb~+ cell lines, consequently effect the transcription of gene. The effect of TSA reply on the length of time. Part IV Analysis of the correlation between the p16-Rb-HDAC1-E2F1 proteins and clinicopathological factor in the gallbladder carcinoma
Thesis 6 Analysis of the correlation between the p16-Rb-HDAC1-E2F1 proteins and clinicopathological factor in the gallbladder carcinoma
Objective To study the characteristics of clinicopathology of gallbladder carcinoma and the expression and significance of p16-Rb-HDAC1-E2F1 proteins in gallbladder carcinoma.
Methods
1. Analysis 65 cases of gallbladder carcinoma in Tongji hospital of Huazhong university of science and technology retrospectively (1991-2005).
2. Research the expression and characteristics of p16-Rb-HDAC1-E2F1 proteins respectively existing in tissues of gallbladder carcinoma, gallbladder adenoma, and chronic cholecystitis by immunehistochemistry.
Results
1. The incidence of coexistence of gallstones in gallbladder carcinoma was 44.64%(25/56). The incidence of hepatitis B in gallbladder carcinoma was 26.79%(15/56). The incidence of schistosome in gallbladder carcinoma was 30.36%(17/56). The incidence of multiple pregnancies in gallbladder carcinoma was 72.98% (27/37). Gallbladder carcinoma accounted for 0.64%(56/8807) of cholecystectomy. Preoperative mis-diagnose were often the gallstone, adenoma and liver cancer. Diagnostic rates of B-ultrasound, CT and MRI were 42. 3% (22/52), 57. 1%[ (14-6) / (20-6) ]and 50%[ (5-2) / (8-2) ] respectively. The γ -GT were all positive in gallbladder carcinoma(4/4).
2. The position of the primary carcinoma of gallbladder located in the collum of gallbladder was 29.73%(11/37), in body of gallbladder was 29.73%(11/37), in fundus of gallbladder was 16.22%(6/37). Pathologic histology type of gallbladder carcinoma included adenocarcinoma (83.93%, 47/56), adenoma malignant progression (7.14%, 4/56), squamous cell carcinoma(5.36%, 3/56) and undifferentiated carcinoma (3.57%, 2/56). The incidence of gallbladder carcinoma in Nevin I stage was 14.29% (8/56), IV stage was 14.29%(8/56), V stage was 10.71%(6/56). There were not significant correlation among sex,the primary position and malignant degree of the gallbladder carcinoma. Liver was often infiltrated by tumor(14/56). Nerve bundle of gallbladder wall were infiltrated by two of them.
3. The positive rates of pl6 and Rb proteins in gallbladder carcinoma tissue are respectively higher than that in adenoma tissue and cholecystitis tissue.The positive rates of HDAC1 and E2F1 proteins in gallbladder carcinoma tissue are respectively higher than that in adenoma tissue and cholecystitis tissue. The positive rate of pl6-Rb-HDAC1-E2F1 proteins in gallbladder carcinoma tissue has no relationship with the differentiation degree of gallbladder carcinoma, Nevin stage, age, gender and gallstone. The expression of pl6 in gallbladder carcinoma infected with hepatitis B virus was 27. 7%, and 55. 6% in the others, there was significant different between them(P<0. 05). It took on some extent positive correlation between the expression of pl6 and the expression of Rb in gallbladder carcinoma( γ=0. 6091, t =4. 48, P<0. 001), it took on some extent negative correlation between the expression of pl6 and the expression of HDAC1 in gallbladder carcinoma( γ=-0. 5215, t=3.56, P<0. 005), it took on some extent negative correlation between the expression of Rb and the expression of HDAC1 in gallbladder carcinoma ( γ=-0.3953, t=2.51, P<0. 05).
Conclusions
1. Gallstones, hepatitis B, schistosome and multiple pregnancies are risk factors of gallbladder carcinoma. The γ -GT may be potential tumor marker.
2. Gallbladder carcinoma often locates in collum and body of gallbladder, pathologic histology type of gallbladder carcinoma is often adenocarcinoma, tumor can infiltrate along nerve bundle of gallbladder wall. There are not significant correlation among sex, the primary position and malignant degree of the gallbladder carcinoma.
3. The expression of p16-Rb-HDAC1-E2F1 proteins in gallbladder carcinoma tissue has no relationship with the differentiation degree of gallbladder carcinoma, Nevin stage, age, and gender.
4. The expression of p16 protein in gallbladder carcinoma tissue is significantly correlative with hepatitis B.
5. The down-regulating expression of p16 protein and Rb protein and down-regulating expression of HDAC1 protein and E2F1 protein is relative with the gallbladder carcinoma pathological characteristics, there are interaction among p16 protein, Rb protein and HDAC1 protein of the gallbladder carcinoma. Thesis 7 Analysis of the correlation between the p16-Rb-HDAC1-E2F1 proteins and clinicopathological factor in the unsuspected gallbladder carcinoma
Objective To study the characteristics of clinicopathology in unsuspected gallbladder carcinoma and the expression and significance of p16-Rb-HDAC1-E2F1 proteins in unsuspected gallbladder carcinoma.
Methods Analysis 23 cases of unsuspected gallbladder carcinoma in Tongji hospital retrospectively, characteristics of clinicopathology were compared with preoperative diagnosed gallbladder carcinoma in same period.Research the expression and characteristics of p16-Rb-HDAC1-E2F1 proteins respectively existing in tissues of unsuspected gallbladder carcinoma by immunehistochemistry.
Results The incidence of coexistence of gallstones in unsuspected gallbladder carcinoma was significantly higher than in preoperative diagnosed gallbladder carcinoma(P<0. 01). The incidence of hepatitis B in unsuspected gallbladder carcinoma was 21. 74%(5/23), in preoperative diagnosed gallbladder carcinoma was 30. 30%(10/33). The incidence of schistosome in unsuspected gallbladder carcinoma was 39. 13%(9/23), in preoperative diagnosed gallbladder carcinoma was 24.24%(8/33). The incidence of multiple pregnancies in unsuspected gallbladder carcinoma was 56. 52%(13/23), in preoperative diagnosed gallbladder carcinoma was 42. 42%(14/33). The γ-GT were all positive in unsuspected gallbladder carcinoma(2/2) and preoperative diagnosed gallbladder carcinoma(2/2). The position of the primary carcinoma of unsuspected gallbladder carcinoma ofen located in the collum and body of the gallbladder, the position of the primary carcinoma of preoperative diagnosed gallbladder carcinoma ofen located in the body and fundus of the gallbladder. The incidence of Nevin I stage and II stage in unsuspected gallbladder carcinoma was significantly higher than that in preoperative diagnosed gallbladder carcinoma(P<0. 05). The incidence of Nevin IV stage in unsuspected gallbladder carcinoma was significantly lower than that in preoperative diagnosed gallbladder carcinoma(P<0. 05). The incidence of Nevin V stage in unsuspected gallbladder carcinoma was significantly lower than that in preoperative diagnosed gallbladder carcinoma(P<0. 01). There was not significant difference between expression of p16-Rb-HDAC1-E2F1 proteins of unsuspected gallbladder carcinoma and expression of p16-Rb-HDAC1-E2F1 proteins of preoperative diagnosed gallbladder carcinoma.
Conclusions Unsuspected gallbladder carcinoma is different from preoperative diagnosed gallbladder carcinoma in the position of the primary carcinoma, the malignant degree and the incidence of coexistence of gallstones. Gallstones, hepatitis B , schistosome and multiple pregnancies are risk factors of unsuspected gallbladder carcinoma. Unsuspected gallbladder carcinoma has similar molecular biological characteristics with preoperative diagnosed gallbladder carcinoma. Ingenuity in this study:
1. The protein signals of p16-Rb-HDAC1-E2F1 in biliary tract cancer involved in the inactivation of antioncogene, regulation of cell cycle, histone acetylation activation of gene promoter, epigenetics, and so on, which was representative in tumorigenesis. The research methods we used have combined these aspects, which apply a available pattern in studying the pathogenesis of cancer.
2. Our study displayed that Rb protein, HDAC1 protein and E2F1 protein can make the specific interaction in biliary tract cancer, and there are the indirect pathways among p16 protein signals and the others, the effect of pl6 in biliary tract cancer rely on the function of Rb, which indicates that p16, Rb, E2F1 and HDAC1 constitute a protein signal pathway.
3. Our study connects the protein signal conduction with the epigenetics, which may clarify the regulation of gene even more. Our study display that the epigenetics can influence cell differentiation by regulation of gene, and the change of the epigenetics connects with protein signals.
4. To extrahepatic cholangiocarcinoma and gallbladder carcinoma, the rate of excision is low and the sensitivity to chemotherapy and actinotherapy is poor. We study the effect of histone deacetylase inhibitor on the growth of biliary tract cancer in vivo and in vitro based on the previous datas of Shengquan Zou' group, which was valuable experiment in biological treatment of biliary tract cancer.
5. The previous datas of Shengquan Zou' group indicate that hepatitis
virus may influence the expression of gene in cholangiocarcinoma. Our research indicates that the expression of pl6 protein in gallbladder carcinoma is significantly correlative with hepatitis B.
引文
1 Albert B. Lowenfels, Patrick Maisonneuve. Epidemiology of biliary tract cancer: what is known, what is unknown?Biliary Tract Cancer International Workshop, 2005: 12
2 Zou SQ, Zhang L, Zen GZ. Clinical epidemiologic characteristics of 430 cases of gallbladder cancer. Chin Med J, 1998,111(5): 391-393.
3 Zou SQ, Zhang L. Relative risk factors analysis of 3922 cases of gallbladder cancer. China J Surg, 2000, 38(11): 805-808.
4 黄志强．临床研究与新世纪胆道外科．第11届全国胆道外科学术会议论文汇编，2004，17-19．
5 邹声泉．胆道肿瘤．见：邹声泉，龚建平主编．外科学—前沿与争论，第一版．北京：人民卫生出版社，2003：600-607．
6 Vogelstein B, Kinz]er KW. Cancer genes and the pathways they control. Nat Med, 2004,10(8): 789-799.
7 邹声泉，高戈，唐启彬，等．肝外胆管癌的细胞分子遗传学研究．第11届全国胆道外科学术会议论文汇编，2004，46-49．
8 唐启彬，石雪涛，邹声泉，等．肝外胆管癌中染色体9p21区段抑癌基因簇表达异常的研究．肿瘤防治研究，2004，31(9)：523-525．
9 Zhao Y, Tan J, Zhuang L, et al. Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci USA. 2005 Nov 1; 102(44): 16090-16095.
10 Ohtani N, Yamakoshi K, Takahashi A, et al. The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression. J Med Invest, 2004 Aug; 51(3-4): 146-153.
1 Liu H, Dibling B, Spike B, et al. New roles for the RB tumor suppressor protein. Curr Opin Genet Dev. 2004 Feb; 14(1): 55-64.
2 Dasgupta P, Betts V, Rastogi S, et al. Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma protein: novel links between apoptotic signaling and cell cycle machinery. J Biol Chem. 2004 Sep 10; 279(37): 38762-9.
3 Mittnacht S. The retinoblastoma protein—from bench to bedside. Eur J Cell Biol. 2005 Mar; 84(2-3): 97-107.
4 Larsen CJ. pRB, p53, p16INK4a, senescence and malignant transformation. Bull Cancer. 2004 May; 91(5): 399-402.
5 Alland L, Muhle R, Hou HJ, et al. Role for N2CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature, 1997,387: 49.
6 Wang ZM, Yang H,Livingston DM. Endogenous E2F-1 promotes timely Go exit of resting mouse embryo fibroblasts. Proc Natl Acad Sci USA, 1998,95 (26): 15583-15586.
7 Simon B, Lubomierski N. Implication of the INK4a/ARF locus in gastroenteropancreatic neuroendocrine tumorigenesis. Ann N Y Acad Sci. 2004 Apr; 1014: 284-99.
1 Zhu L. Tumour suppressor retinoblastoma protein Rb: A transcriptional regulator. Eur J Cancer. 2005 Nov; 41(16):2415-27.
2 Niu Y, Li Y, Niu RF, et al. Correlation of E2F-1 and Rb expression with papillomatosis and ductal carcinoma in situ of the breast. Zhonghua Zhong Liu Za Zhi. 2004 May;26(5) :290-3.
3 Zhong M, Wang J, Zhang B, et al. Expression of pRb and E2F-1 and telomerase activity in ameloblastoma. Zhonghua Kou Qiang Yi Xue Za Zhi.2004 Sep; 39(5) :406-9.
4 Rogoff HA, Kowalik TF. Life, death and E2F:linking proliferation control and DNA damage signaling via E2F1. Cell Cycle. 2004 Jul; 3(7):845-6.
5 Pickering MT, Kowalik TF. Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene. 2005 Sep 26; [Epub ahead of print]
6 Jiang Y, Saavedra HI, Holloway MP, Leone G, Altura RA. Aberrant regulation of survivin by the RB/E2F family of proteins.J Biol Chem. 2004 Sep 24;279 (39): 40511-20.
7 Dasgupta P, Betts V, Rastogi S, et al.Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma protein: novel links between apoptotic signaling and cell cycle machinery. J Biol Chem. 2004 Sep 10;279(37):38762-9.
8 Gonzalo S, Blasco MA. Role of Rb family in the epigenetic definition of chromatin. Cell Cycle. 2005 Jun;4(6):752-5.
9 Luo RX, Postigo AA, Dean DC. Retinoblastoma interacts switch histone deacetylase to repress transcription. Cell, 1998, 92:496-473.
10 Magnaghi JL, Groisman R,Nagulbneva L, et al.Retinoblastoma protein repress transcription by recruiting a histone deacetyklase. Nature, 1998, 391(6667):601-605.
1 Dasgupta P, Betts V, Rastogi S, et al. Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma protein: novel links between apoptotic signaling and cell cycle machinery. J Biol Chem. 2004 Sep 10; 279(37): 38762-9.
2 Mittnacht S. The retinoblastoma protein—from bench to bedside. Eur J Cell Biol. 2005 Mar; 84(2-3): 97-107.
3 Larsen CJ. pRB, p53, p16INK4a, senescence and malignant transformation. Bull Cancer. 2004 May; 91(5): 399-402.
4 Alland L, Muhle R, Hou HJ, et al. Role for N2CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature, 1997, 387: 49.
5 Wang ZM, Yang H, Livingston DM. Endogenous E2F-1 promotes timely Go exit of resting mouse embryo fibroblasts. Proc Nat] Acad Sci USA, 1998,95 (26): 15583-15586.
6 Simon B, Lubomierski N. Implication of the INK4a/ARF locus in gastroenteropancreatic neuroendocrine tumorigenesis. Ann N Y Acad Sci. 2004 ipr; 1014: 284-99.
7 Claudio PP, DeLuca A, Howard CM, et al. Function alanalysis of pRb/p130 interaction with cyclins. Cancer Res, 1996, 56(9): 2003-2008.
8 He XS, Rong YH, Su Q, et al. Expression of p16 gene and Rb protein in gastric carcinoma and their clinicopathological significance. World J Gastroenterol. 2005 ipr 21; 11(15): 2218-2223.
1 Ikeguchi M, Oi K, Hirooka Y, Kaibara N. CD8+ lymphocyte infiltration and apoptosis in hepatocellular carcinoma. Eur J Suyg Oncol 2004; 30: 53-57
2 Dangles V, Validire P, Wertheimer M, Richon S, Bovin C, Zeliszewski D, Vallancien G, Bellet D. Impact of human bladder cancer cell architecture on autologous T-lymphocyte activation. Int J Cancer 2002; 98: 51-56
3 Zhao Y, Tan J, Zhuang L, et al. Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci USA. 2005 Nov 1; 102(44): 16090-5.
4 Abrahams VM, Kamsteeg M, Mot G. The Fas/Fas ligand system and cancer: immune privilege and apoptosis. Mol Biotechnol 2003; 25: 19-30
5 Griffith TS, Ferguson TA. The role of FasL-induced apoptosis in immune privilege. Imunol Today 1997; 18: 240-244
6 Humbert PO, Verona R, Trimarchi JM, et al. E2f3 is critical for normal cellular proliferation Genes Dev, 2000,14(6): 690-703.
7 Dimri GP, Itahana K, Acosta M, et al. Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14 (ARF) tumor suppressor. Mol Cell Biol, 2000, 20(1): 2732 285.
8 Frolov MY, Dyson NJ. Molecular mechanisms of E2F-dependent activation and pRB-mediated repression. J Cell Sci. 2004 May 1;117(Pt 11): 2173-81.
9 Pennaneach V, Barbier V, Regazzoni K, et al. Rb inhibits E2F-1-induced cell death in a LXCXE-dependent manner by active repression. J Biol Chem. 2004 May 28; 279(22): 23376-83.
1 Jenuwein T, Allis CD. Translating the histone code. Science, 2001, 203(5532): 1074-1080.
2 Geiman TM, Robertson KD. Chromatin remodeling, histone modifications, and DNA methylation-how does it all fit together? J Cell Biochem. 2002,87(2): 117-125.
3 Redner RL, Chen JD, Rush EA, et al. The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties. Blood. 2000,95(8): 2683-2690.
4 Richards EJ, Elgin SC. Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell. 2002,108(4): 489-450.
5 Luo RX, Postigo AA, Dean DC. Retinoblastoma interacts switch histone deacetylase to repress transcription. Cell, 1998, 92: 496-473.
6 Magnaghi JL, Groisman R, Nagulbneva h, et al. Retinoblastoma protein repress transcription by recruiting a histone deacetyklase. Nature, 1998, 391(6667): 601-605.
1 邹声泉，徐立宁．重视意外胆囊癌的诊治问题．中华外科杂志，2005，43(13)：833-5．
2 徐立宁，邹声泉．胆囊癌分子生物学研究现状及前景．实用肿瘤杂志，2005， 20(1)：7-9．
3 (美)萨比斯通，主编；王德炳，主译．克氏外科学，第十五版[M]．北京：人民卫生出版社，2000：15-32．
4 黄志强．胆道肿瘤．见：吴阶平，裘法祖主编．黄加驷外科学，第六版．北京：人民卫生出版社，2000：1290-3．
5 刘小方，邹声泉，裘法祖．肝炎病毒感染与肝门部胆管癌发病关系的探讨．中华外科杂志，2002，40(6)：420-422．
6 石景森．走出胆囊癌诊断的思维误区．临床外科杂志，2006，14(2)：68-69．
7 刘绍诰．胆囊癌．见：黄志强主编．当代胆道外科学，第一版．上海：上海科学技术文献出版社，1998：607-14．
8 Korenjak M, Brehm A. E2F-Rb complexes regulating transcription of genes important for differentiation and development. Curr Opin Genet Dev. 2005 Oct; 15(5): 520-7.
9 范祖森，敖世洲．肿瘤抑制基因RB与细胞周期调控研究新进展．生物化学与生物物理进展，1999，26(5)：429-433．
10 Zhu L. Tumour suppressor retinoblastoma protein Rb: A transcriptional regulator. Eur J Cancer. 2005 Nov; 41(16): 2415-27.
11 Niu Y, Li Y, Niu RF, et al. Correlation of E2F-I and Rb expression with papillomatosis and ductal carcinoma in situ of the breast. Zhonghua Zhong Liu Za Zhi. 2004 May; 26(5): 290-3.
12 Zhong M, Wang J, Zhang B, et al. Expression of pRb and E2F-1 and telomerase activity in ameloblastoma. Zhonghua Kou Qiang Yi Xue Za Zhi. 2004 Sep; 39(5): 406-9.
13 Rogoff HA, Kowalik TF. Life, death and E2F: linking proliferation control and DNA damage signaling via E2F1. Cell Cycle. 2004 Jul; 3(7): 845-6.
14 Pickering MT, Kowalik TF. Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene. 2005 Sep 26; [Epub ahead of print]
15 Jiang Y, Saavedra HI, Holloway MP, Leone G, Altura RA. Aberrant regulation of survivin by the RB/E2F family of proteins. J Biol Chem. 2004 Sep 24; 279(39): 40511-20.
16 Luo RX, Postigo AA, Dean DC. Retinoblastoma interacts switch histone deacety]ase to repress transcription. Cell, 1998, 92: 496-473.
17 Magnaghi JL, Groisman R, Nagulbneva L, et al. Retinoblastoma protein repress transcription by recruiting a histone deacetyklase. Nature, 1998, 391(6667): 601-605.
18 Pennaneach V, Barbier V, Regazzoni K, et al. Rb inhibits E2F-1-induced cell death in a LXCXE-dependent manner by active repression. J Biol Chem. 2004 May 28;279(22): 23376-83.
19 Zhao Y, Tan J, Zhuang L, et al. Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci USA. 2005 Nov 1; 102(44): 16090-5.
20 Narimatsu T, Tamori A, Koh N, et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology, 2004; 47(1): 26-31.
21 Wang ZW, Peng ZH, Li K, et al. Effect of p16 gene on carcinogenesis of hepatitis B virus related hepatocellular carcinoma. Zhonghua Zhong Liu Za Zhi, 2003 Jul; 25(4): 356-8.
22 刘小方，邹声泉．肝门部胆管癌中乙肝、丙肝病毒感染与p53异常表达的关系及意义．中华肝胆外科杂志，2003，9(3)：177-181．
23 Zou SO, Zhang L, Zen GZ. Clinical epidemiologic characteristics of 430 cases of gallbladder cancer. Chin Med J, 1998, 111(5):391-3.
24 Zou SQ, Zhang L. Relative risk factors analysis of 3922 cases of gallbladder cancer. China J Surg, 2000,38(11): 805-8.
25 邹声泉．胆道肿瘤．见：邹声泉，龚建平主编．外科学—前沿与争论，第一版．北京：人民卫生出版社，2003-600-7．
1 邹声泉，徐立宁．重视意外胆囊癌的诊治问题．中华外科杂志，2005，43(13)：833-5．
2 MARCIAL-ROJAS RA, MEDINA R. Unsuspected carcinoma of the gallbladder in acute and chronic cholecystitis. Ann Surg, 1961, 153: 289-98.
3 田华，陈力，刘桂杰，等．意外胆囊恶性肿瘤的外科治疗．中华外科杂志，2005，43(13)：836-8．
4 Zou SQ, Zhang L, Zen GZ. Clinical epidemiologic characteristics of 430 cases of gallbladder cancer. Chin Med J, 1998,111(5): 391-3.
5 Zou SQ, Zhang L. Relative risk factors analysis of 3922 cases of gallbladder cancer. China J Surg, 2000, 38(11): 805-8.
6 邹声泉．胆道肿瘤．见：邹声泉，龚建平主编．外科学—前沿与争论，第一版．北京：人民卫生出版社，2003：600-7．
1 Korenjak M, Brehm A. E2F-Rb complexes regulating transcription of genes important for differentiation and development. Curr Opin Genet Dev. 2005 Oct;15(5): 520-527.
2 Liu H, Dibling B, Spike B, et al. New roles for the RB tumor suppressor protein. Curr Opin Genet Dev. 2004 Feb; 14(1):55-64.
3 Sparkes MS, Sparkes MC, Willson MG, et al. Regional assignment of genes for human esterase D and retinoblastoma to chromosome band 13q14. Science, 1980, 208: 1042-1044.
4 Bookstein R, Lee H. Human retinobl astoma susceptibility gene: genomic organization and analysis of hetterozygous intragenic deletins mutants. Proc Natl Acad Sui USA, 1988, 85: 2210-2114.
5 Hong FD, Huang H, et al. Structure of the human retinoblastoma gene. Proc Natl Acad Sci USA, 1989, 86: 5502-5506.
6 黄倩，汪健，陶勇浩，等．正常人及视网膜母细胞瘤患者Rh基因启动于结构、功能和突变的研究．中华肿瘤，1998，20(3)：165-167．
7 Sowa Y, Shiioy, Fujita T, et al. Retinoblastoma binding factorl site in the core promotor region of the human retinoblastoma gene is activated byb GABP/ E4TF1.Cancer Res, 1997,57: 3145-3148.
8 Friend SH, Bernards R, Rogelj S, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature, 1986, 323: 643.
9 Lee WH, Bookatein R, tiong F, et al. Human retinoblastoma suscepitibility gene, cloning, identification and sequence. Science, 1987, 235: 1394.
10 Fung YKT, Murphree AL, T'Ang A, et al. Struetural evidence for the authenticity of the human recinoblastoma gene. Science, 1987,236: 1657.
11 Landschulze WH, Johnson PF, Mcknight SL. Tbe leucine zipper:a hypothetical structure common to a new class of DNA binding proteins. Science, 1988, 240: 1759-1763.
12 Ritety DJ, Liu CY, Lee WH, et al. Mutations of N-terminal regions renders the Retinoblastoma protein in sufficient functions in development and tumor suppression. Mol Col Biol,1997,17: 7342-7352.
13 Smith RC, Witts KN, Antelman M, et al. Adenovirial constructs econding phosphorylation competent full length and truncated forma of the human retinoblatoma protein inhibit myocyte proliferation and neointima formation. Circulation, 1997,96: 1899-1905.
14 Borges HL, Bird J, Wasson K, et al. Tumor promotion by caspase-resistant retinoblastoma protein. Proc Natl Acad Sci USA. 2005 Oct 25; 102(43): 15587-15592.
15 Dasgupta P,Betts V, Rastogi S, et al.Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma proteinrnovel links between apoptotic signaling and cell cycle machinery. J Biol Chem. 2004 Sep 10;279(37):38762-38769.
16 Ludlow JW, DeCaprio JA , Huang CM, et al. SV 40 large T antigen binds prefertially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell, 1989, 56:57-65.
17 Nishinaka T, Fu YH, Chen LI, et al.A unique cathepsin-like protase isolated from CV1 cells is involved in rapid degradation of retinoblastoma susceptibility gene product, RB, and transcription factor SPl.Biochim Biophys Acta, 1997, 1351:274-286.
18 Mittnacht S. The retinoblastoma protein—from bench to bedside.Eur J Cell Biol. 2005 Mar;84(2-3):97-107.
19 Larsen CJ. pRB, p53, pl6INK4a, senescence and malignant transformation. Bull Cancer.2004 May;91(5):399-402.
20 Angus SP, Mayhew CN, Solomon DA, et al. RB reversibly inhibits DNA replication via two temporally distinct mechanisms. Mol Cell Biol. 2004 Jun;24(12):5404-5420.
21 Mayhew CN, Perkin LM, Zhang X, Sage J, Jacks T, Knudsen ES. Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents. Oncogene. 2004 May 20;23(23):4107-4120.
22 Grana X, Reddy EP.Cell cycle control in mammlian cells:role of cyclins, cyclin dependent kinase (CDKs), growth suppressor and cyclin dependent kinase inhibitors (CDKs). J Oncogene, 1995, 11:21-26.
23 DeGregori J. The Rb network. J Cell Sci. 2004 Jul 15;117(Pt 16): 3411-3.
24 Zhu L. Tumour suppressor retinoblastoma protein Rb: A transcriptional regulator. Eur J Cancer. 2005 Nov;41(16):2415-2427.
25 Niu Y, Li Y, Niu RF, et al. Correlation of E2F-1 and Rb expression with papillomatosis and ductal carcinoma in situ of the breast. Zhonghua Zhong Liu Za Zhi. 2004 May; 26(5):290-293.
26 Zhong M, Wang J, Zhang B, et al. Expression of pRb and E2F-1 and telomerase activity in ameloblastoma. Zhonghua Kou Qiang Yi Xue Za Zhi. 2004 Sep; 39(5):406-409.
27 Rogoff HA, Kowalik TF. Life, death and E2F:linking proliferation control and DNA damage signaling via E2F1. Cell Cycle. 2004 Jul;3(7):845-846.
28 Pickering MT, Kowalik TF.Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene. 2005 Sep 26; [Epub ahead of print]
29 Jiang Y, Saavedra HI,Holloway MP, Leone G, Altura RA. Aberrant regulation of survivin by the RB/E2F family of proteins. J Biol Chem. 2004 Sep 24; 279(39):40511-40520.
30 Gonzalo S, Blasco MA. Role of Rb family in the epigenetic definition of chromatin. Cell Cycle. 2005 Jun;4(6):752-755.
31 Hernando E, Nahle Z, Juan G,et al. Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature. 2004 Aug 12; 430 (7001):797-802.
32 McCabe MT, Davis JN, Day ML. Regulation of DNA methyltransferase 1 by the pRb/E2F1 pathway. Cancer Res. 2005 May 1;65(9):3624-3632.
33 Zhao Y, Tan J, Zhuang L, et al.Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci USA. 2005 Nov 1;102(44):16090-16095.
34 Nagy L, Kao HY, Chakravarti D, et al. Nuclear recptor reprerssion mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell, 1997, 89:373.
35 Hassig CA, Fleischer TC, Billin AN et al. Histone deacetylase activity is requied for transcriptionsl repression by mSin3A. Cell, 1997,89:341.
36 Alland L, Muhle R, Hou HJ, et al. Role for N2CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature, 1997, 387:49.
37 Megee PC, Morgan BA, Smith MM. Histone H4 and the maintenance of genome integrity. Genes Dev,1995, 9:1716.
38 Yu X, Guo ZS, Marcu MG, et al. Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228. J Natl Cancer Inst, 2002, 94:504.
39 Liu L T, Chang HC, Chiang LC et al. Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion. Cancer Res, 2003, 63:3069.
40 Tsurutani J, Soda H, Oka M, et al. Antiproliferative effects of the histone deacetylase inhibitor FR901228 on small-cell lung cancer lines and drug-resistant sublines.Int J Cancer, 2003, 104:238.
41 Sakimura R, Tanaka K, Nakatani F, et al. Antitumor effects of histone deacetylase inhibitor on Ewing's family tumors. Int J Cancer. 2005 Sep 20;116(5):784-792.
42 Atadja P, Hsu M, Kwon P, et al. Molecular and cellular basis for the anti-proliferative effects of the HDAC inhibitor LAQ824. Novartis Found Symp. 2004;259:249-266.
43 Luo RX, Postigo AA, Dean DC. Retinoblastoma interacts switch histone deacetylase to repress transcription. Cell, 1998, 92:496-473.
44 Magnaghi JL, Groisman R, Nagulbneva L, et al. Retinoblastoma protein repress transcription by recruiting a histone deacetyklase. Nature, 1998, 391(6667):601-605.
45 Pennaneach V, Barbier V, Regazzoni K, et al. Rb inhibits E2F-1-induced cell death in a LXCXE-dependent manner by active repression. J Biol Chem, 2004 May 28; 279(22):23376-23383.
46 Jenuwein T, Allis CD. Translating the histone code. Science, 2001, 203(5532):1074-1080.
47 Geiman TM, Robertson KD. Chromatin remodeling, histone modifications, and DNA methylation-how does it all fit together? J Cell Biochem. 2002,87(2) : 117-125.
48 Redner RL, Chen JD, Rush EA, et al.The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties. Blood, 2000, 95(8):2683-2690.
49 Richards EJ, Elgin SC. Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell, 2002, 108(4): 489-450.
50 Wang ZM, Yang H, Livingston DM. Endogenous E2F-1 promotes timely G_0 exit of resting mouse embryo fibroblasts.Proc Natl Acad Sci USA, 1998,95 (26):15583-15586.
51 Slansky JE, Farnham PJ, Introduction to the E2F family:protein structure and gene regulation[J]. Curr ToD Microbiol Immunol, 1996,208:1.
52 Moberg KH, Logan TJ, Tyndall WA, et al. Three distinct elements within the murine c-myc promoter are required for transcription [J]. Oncogene, 1992, 7:411-412.
53 Leone G, DeGregori J, Yan Z, et al. E2F3 activity is regulated during the cell cycle and is required for the induction of S phase. Genes Dev, 1998, 12(14): 2120-2130.
54 Humbert PO, Verona R, Trimarchi JM, et al. E2f3 is critical for normal cellular proliferation. Genes Dev, 2000, 14(6):690-703.
55 Dimri GP, Itahana K, Acosta M, et al. Regulation of a senescence checkpoint response by the E2F1 transcription factor and p41(ARF) tumor suppressor. Mol Cell Biol, 2000, 20(1):273-285.
56 Frolov MV, Dyson NJ. Molecular mechanisms of E2F-dependent activation and pRB-mediated repression. J Cell Sci, 2004 May 1;117(Pt 10:2173-81.
57 Ginsberg D. E2F1 pathways to apoptosis. FEBS Lett,2002, 529(1): 122-125.
58 Krek W, Xu G, Livingston DM. Cyclin A-kinase regulation of E2F-1 DNA binding function underlies suppression of a S phase checkpoint. Cell, 1995, 83:1149-1158.
59 Rogoff HA, Pickering MT, Frame FM, et al. Apoptosis associated with deregulated E2F activity is dependent on E2F1 and Atm/Nbs1/Chk2. Mol Cell Biol, 2004 Apr; 24(7):2968-77.
60 Zacharatos P,Kot sinas A, Evangelou K. Distinct expression patterns of t he t ranscription factor E2F-1 in relation to tumour growth parameters in common human carcinomas. J Pat hol, 2004, 203 (3): 744-753.
61 Wyllie AH. E2F1 selects tumour cells for both life and death.J Pathol, 2002, 198(2):139-141.
62 Yang XH, Siadek TL. Overexpression of the E2F-1 transcription factor gene mediates cell transformation. Gene Expr, 1995,4:195-204.
63 Hunt KK, Deng J, Liu TJ, et al. Adenovirus mediated overexpression of the transcription factor E2F-1 induces apoptosis in human breast and ovarian2carcinoma cell lines and does not require P53. Cancer Res,1997, 57:4722-4726.
64 Balasubramanian S, Ahmad N, Mukhtar H. Upregulation of E2F transcription factors in chemically induced mouse skin tumors. Int J Oncol, 1999, 15(2) :387.
65 Pierce AM, Fisher SM, Conti CJ, et al. Deregulated expression of E2F1 induces hyperplasia and cooperates with ras in skin tumor development. Oncogene, 1998, 16(10):1267.
66 Dicker AJ, Popa C, Dahler AL, et al. E2F-1 induces proliferation-specific genes and suppresses squamous differenuiation-specific genes in human epidermal keratinocytes. Oncogene, 2000, 19(25) :2887.
67 Yamazaki K, Hasegawa M, Ohoka I, et al. Increased E2F-1 expression via tumour cell proliferation and decreased apoptosis are correlated with adverse prognosis in patients with squamous cell carcinoma of the oesophagus. J Clin Pathol, 2005 Sep;58(9):904-10.
68 Yamasaki L, Jacks T, Bronson, et al. Tumor induction and tissue atrophy in mice lacking E2F-1. Cell, 1996, 85:537-548.
69 Gorgoulis VG, Zacharatos P, Mariatos G, et al. Transcription factor E2F-1 act s as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas. J Pathol, 2002, 198(2): 142-156.
70 Eymin B, Gazzeri S, Brambilla C, et al. Distinct pattern of E2F1 expression in human lung tumours:E2F1 is upregulated in small cell lung carcinoma. Oncogene, 2001, 20(14):1678-1687.
71 DeMuth JP, Jackson CM, Weaver DA, et al. The gene expression index c-myc x E2F-1/p21 is highly predictive of malignant phenotype in human bronchial epit helial cells. Am J Respir Cell Mol Biol, 1998, 19(1): 18-24.
72 Yamasaki L. Balancing proliferation and apoptosis in vivo:the Goldilocks theory of E2FPDP action. Biochim Biophys Acta, 1999, 1423:M9-15.
73 Halaban R. Rb/E2F:a two-edged sword in the melanocytic system. Cancer Metastasis Rev, 2005 Jun24(2): 339-356.
74 O' Conner DJ, Lu X. Stress signals induce transcriptionally inactive E2F-1 independently of p53 and Rb. Oncogene, 2000,19(20): 2369-2376.
75 McCabe MT, Azih OJ, Day ML. pRb-Independent growth arrest and transcriptional regulation of E2F target genes. Neoplasia, 2005 Feb; 7(2): 141-51.
76 Thomas DW. Cellular senescence and cancer. J Pathology, 1999, 187: 100-111.
77 Claudio PP, DeLuca A, Howard CM, et al. Function alanalysis of pRb/p130 interaction with cyclins. Cancer Res, 1996,56(9): 2003-2008.
78 He XS, Rong YH, Su Q, et al. Expression of p16 gene and Rb protein in gastric carcinoma and their clinicopathological significance. World J Gastroenterol. 2005 Apr 21; 11(15): 2218-2223.
79 范祖森，敖世洲．肿瘤抑制基因RB与细胞周期调控研究新进展．生物化学与生物物理进展，1999，26(5)：429-433．
80 Ohlson LC, Koroxenidou L, Porsch-Hallstrom I. Mitoinhibitory effects of the tumor promoter 2-acetylaminofluorene in rat liver: loss of E2F-1 and E2F-3 expression and cdk 2 kinase activity in late G1. J Hepatol, 2004 Jun; 40(6): 957-62.
81 Serrano M, Hannon GJ, Beach D.A new regulatory motif in cell-cycle control causing specific inhibition of cyclinD/CDK4. Nature, 1993, 366(6456): 704-707.
82 Kamb A, Shattuck, Eeles R, et al. Ana]ysis of the P16 gene (CDKN2) usa candidate for the chromosome 9p melanoma susceptibility cocas. Science, 1994,264:436.
83 Sherr CJ. Mammalian G1 cyclin. Cell, 1993,73(6): 1059-1065.
84 Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 1994, 264(5157):436-440.
85 Simon B, Lubomierski N. Implication of the INK4a/ARF locus in gastroenteropancreatic neuroendocrine tumorigenesis. Ann N Y Acad Sci, 2004 Apr;1014:284-299.
86 Sharpless NE. INK4a/ARF: a multifunctional tumor suppressor locus. Mutat Res, 2005 Aug 25; 576(1-2):22-38.
87 Marx J . New tumor suppressor may rival p53. Science, 1994, 264(5157):344-345.
88 Schulze A , Zerf ass K, Spit kovsky D , et al . Activation of the E2F transcription factor by cyclin D1 is blocked by pl6INK4, the product of the putative tumor suppressor gene MST1. Ocogene, 1994, 9(12):3475-3482.
89 Nilsson K, Landberg G. Subcellular localization, modification and protein complex formation of the cdk-inhibitor p16 in Rb-functional and Rb-inactivated tumor cells. Int J Cancer, 2005 Sep 13; [Epub ahead of print]
90 Plath T, Detjen K, Welzel M, et al.A novel function for the tumor suppressor pl6INK4a: induction of anoikis via upregulation of the α 5β1 fibronectin receptor. J Cell Biol, 2000, 150(6) : 1467-1478.
91 Nobori T, Miura K, Wu DJ, et al. Deletions of the cycle dependent kinase-4 inhibitor gene in multiple human cancers. Nature, 1994, 368(6473):753-756.
92 Okamoto A, Demetrick DJ, Spillare EA, et al. Mutations and altered expression of pl6INK4 in human cancer. Proc Natl Acad Sci USA, 1994, 91(23): 11045-11049.
93 Xavier G. Cell cycle control in mammalian cells:role of cyclins, cyclin dependent kinases(CDKs), growth suppressor genes and cyclin dependent kinase inhibitors(CKIs). Oncogene, 1995, 11:211-219.
94 Liggett WH Jr, Sidransky D. Role of the pl6 tumor suppressor gene in cancer. J Clin Oncol, 1998, 16(3):1197-1206.
95 Shapiro GI, Edwards CD, Kobzik L, et al. Reciprocal Rb in activation and p16INK4 expressionin primary lung cancers and cell lines. Cancer Res, 1995, 55:505-509.
96 Ohtani N, Yamakoshi K,Takahashi A, et al.The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression. J Med Invest,2004 Aug;51(3-4):146-153.
97 Serrano M, Grogory J, Hannon, et al. A new regulatory motif in cellcycle control causing specific inhibition of cycling D/cdk4. Nature, 1993, 366:704.
98 Noborl T,Kaoru Mlura, David J, et al.Deletions of the cycling dependent kinase 4 inhibitor hence in multiple human cancers. Nature, 1994,368:753.
99 Cairns P, Polascik TJ, Eby Y, et al. Frequency of homozygous deletion at P16 CKDN2 in primary tumors. Nature Genetics,1995, 11:210-212.
100 Volgareva GM, Zavalishina LE, Andreeva IuIu, et al. Expression of pl6INK4a in various cancer cells. Arkh Patol, 2004 Sep-Oct ;66(5) :3-5.
101 Nilsson K, Svensson S, Landberg G. Retinoblastoma protein function and p16INK4a expression in actinic keratosis, squamous cell carcinoma in situ and invasive squamous cell carcinoma of the skin and links between pl6INK4a expression and infiltrative behavior. Mod Pathol, 2004 Dec;17(12):1464-1474.
1 (美)萨比斯通，主编；王德炳，主译．克氏外科学，第十五版[M]．北京：人民卫生出版社，2000：15-32．
2 石景森．我国胆囊癌的发病情况及外科处理[J]．肝胆胰外科杂志，1999， 11(1)：51-52．
3 Merimsky O, Issakov J, Schwartz I, et al. Lack of ErbB-2 oncogene product overexpression in soft tissue sarcomas[J]. Acta Oncol, 2002, 41(4): 366-368.
4 Kamel D, Paakko P, Nuorva K, et al. p53 and c-erbB-2 protein expression in adenocarcinomas and epithelial dysplasias of the gallbladder[J]. J Pathol, 1993, 170(1): 67-72.
5 Chow NK, Huang SM, Chang SH. Significance of C-erbB-2 expression normal and neoplastic epithelium of biliary tract[J]. Anticancer Res, 1995, 15: 1055.
6 Akisik E, Bavbek S, Dalay N. CD44 variant exons in leukemia and lymphoma[J]. Pathol Oncol Res, 2002, 8(1): 36-40.
7 Gunthert U, Hofmann M, Rudy W, et al. A new variant of glycoprotein CD44confersmetastasis potential to rat carcinomacells[J]. Cell, 1991, 65: 13-24.
8 谷化平，尚培中，刘艳茹．原发性胆囊癌CD44v6和Bcl-2的表达及其与临床病理的联系[J]．中国普外基础与临床杂志，2003，10(1)：46-48．
9 Steeg PS, Bevilacqua G, Kopper L, et al. Evidence for a novel gene associated witha low tumor metastatic potential[J]. J Natl Cancer Inst, 1988, 80(17): 204-208.
10 吴晓华，沈忠英，汤雪明．肿瘤转移抑制基因rim23研究进展[J]．生命科学，1996，8(3)：18-21．
11 Hennessy C, Henry JA, May FE, et al. Expression of the antimetastatic gene nm23 in human breast cancer: an association with good prognosis[J]. J Natl Cancer Inst, 1991, 83(4): 281-284.
12 Galigo MA, Cipollini G, Berti A, et al. rim23 gene expression in human advanced phase of tumor progression[J]. Int Cancer, 1997, 74(1): 102-107.
13 Huang G, Song Y, He G. mRNA expression and mutation of MTA1 and nm23H1 genes in ovarian carcinoma in relation to lymph node metastasis[J]. Zhonghua ZhongLiu ZaZhi., 2001, 23(1): 31-34.
14 Galani E, Sgouros J, Petropoulou C, et al. Correlation of MDR-1, nm23-H1 and H Sema E gene expression with histopathotogical findings and clinical outcome in ovarian and breast cancer patients[J]. Anticancer Res, 2002, 22(4): 2275-2280.
15 Forte A, D'Urso A, Gallinaro LS, et al. NM23 expression as prognostic factor in colorectal carcinoma[J]. G Chir, 2002, 23(3): 61-63.
16 Belev B, Aleric I, Vrbanec D, et al. Nm23 gene product expression in invasive breast cancer-immunohistochemical analysis and clinicopathological correlation[J]. Acta Oncol, 2002, 41(4): 355-361.
17 谷化平，尚培中，刘艳茹．nm23-H1和E-Cadherin基因蛋白在胆囊癌组织中的表达[J]．中国现代普通外科进展，2002，5(1)：29-31．
18 邹声泉．胆囊癌的分子生物学研究现状和进展[J]．世界华人消化杂志，1999，7(11)：926-927．
19 Imam M, Hoshi T, Ogawa K. K-ras codon 12 mutations in biliary tract tumors detected by polymerase chain reaction denaturing gradient gel ectrophrosis[J]. Cancer, 1994, 73: 2727.
20 Ajiki T, Fujimor H, Onoyyama M. K-ras gene mutation in gallbladder carcinoma and dysplasia[J]. Gut, 1996, 38: 426-429.
21 Matsubara T, Sakurai Y, Sasyama Y. K-ras point mutation in cancerous and noncancerous biliary epithelium in patient with pancreaticobiliary maljunction[J]. Cancer, 1996, 77: 1752-1757.
22 卿德科，夏亮芳，庞家芳．原发性胆囊癌FaS p21、PCNA、AgNORs的对比研究．中国肿瘤临床，1999，26(1)：59-60．
23 Hahn SA, Schutte M, Hoque AT, et al. DPC4 a candidate tumor suppressor gene at human chromosome 18q21.1[J]. Science, 1996, 19(271): 350-353.
24 Riggins GJ, Kinzler KW, Vogelstein B, et al. Frequency of Smad gene mutations in human cancer[J]. Cancer Res, 1997, 57: 2578-2580.
25 Lagna G, Hata A, Hemmati BA, et al. Partnership between DPC4 and Smad proteins in TGFβ Signaling pathways[J]. Nature, 1996, 383: 832-836.
26 Gustin A, Pederson L, Miller R, et al. Application of molecular biology studies to gene therapy treatment strategies[J]. World J Surg, 2002, 26(7): 854-860.
27 Massague J. TGFβ signaling: receptors, transducers and Mad proteins[J]. Cell, 1996, 85: 947-950.
28 Schutte M. DPC4 gene alterations in human cancer, and their functional implications[J]. Ann Oncol, 1999, 10(4): 56-59.
29 陈军，胡志前，蒋树娟，等．原发性胆囊癌抑癌基因DPC4蛋白表达及其意义[J]．肝胆胰外科杂志，2002，14(1)：52-53．
30 Santoni RE, Duro D, Farkas T, et al. E2Factivity is essential for survival of Myc-overexpressing human cancer cells[J]. Oncogene, 2002, 21(42): 6498-6509.
31 Rim ttS, Shin JY, Ahn DK, et al. Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large W antigen[J]. Exp Mol Med, 2001, 33(4): 293-298.
32 梁刚，沈陶然，徐小宏，等．早期胆囊癌癌基因的表达[J]．临床与实验病理学杂志， 1997，13(4)：333-335．
33 龚非力．医学免疫学，第一版[M]．北京：科学出版社，2000：261．
34 范毓东．Bcl-2与胆管细胞癌[J]．国外医学外科学分册，1999，26(3)：159-161．
35 Tsujimoto Y, Finger LR, Yunis J, et al. Cloning of the chromosome breakpoint neoplastic B cells with the t(14;18) chromosome translocation[J]. Science, 1984, 226(30): 1097-1099.
36 谷化平，尚培中，周翠玲．胆囊癌组织CD15和Bcl-2的表达及相关性研究[J]．临床肝胆病杂志，2001，17(1)：57-58．
37 Itoh N, Tsujimoto Y, Nagata S. Effect of bcl-2 on fas antigen-mediated cell death[J]. J Immunol, 1993, 151(2): 621-627.
38 Mikami T, Yanagisawa N, Baba H, et al. Association of bcl-2 protein expression with gallbladder carcinoma differentiation and progression and its relation toapoptosis[J]. Cancer, 1999, 85(2): 318-325.
39 徐立宁，丁志强．胆囊癌组织Bcl-2蛋白和Fas／FasL蛋白的表达及相关性研究[J]．中国现代医学，2003，13(4)：28-30．
40 Gao Y, FergusonDO, Xie W, et al. Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development[J]. Nature, 2000, 404(6780): 897-900.
41 Difilippantonio M, Petersen S, Chen HT, et al. Evidence for replicative repair of DNA double-strand breaks leading to oncogenie translocation and gene amplification[J]. J Exp Med, 2002, 196(4): 469-80.
42 Matsubara T. K-ras point mutations in cancerous and noncancerous biliary epithelium inpatient with pancreaticobiliary maljunction[J]. Cancer, 1996, 77: 1752.
43 Sarpa A. Neoplasma of the ampwla of vater K-ras and p53 mutations[J], am J Pathol, 1993, 142: 1163.
44 Serrano M, Harmon GJ, Beach DA. A new regulatory motifin cell-cycle control causing specific inhibition of cyclin D/CDK4[J]. Nutune (lond), 1993, 366(6456): 704-707.
45 Kamb A, Gruis NA, Weaver FJ, et al. A cell cycle regulator potentially involved in genesis of many tumor types[J]. Science, 1994, 264(5157): 436-440.
46 Ku JL, Yoon KA, Kim IJ, et al. Establishment and characterisation of six human biliary tract cancer cell lines[J]. Br J Cancer, 2002, 87(2): 187-193.
47 马红兵，狄政莉，王作仁，等．抑癌基因p16、Rb的表达与胆囊癌预后的关系[J]．西安医科大学学报，2001，22(4)-343-345．
1 MARCIAL-ROJAS RA, MEDINA R. Unsuspected carcinoma of the gallbladder in acute and chronic cholecystitis. Ann Surg, 1961, 153: 289-298.
2 Andersson A, Bergdahl L, Zeuchner E. Carcinoma as an unexpected histologic diagnosis in gallbladder disease, gentralbl Chir, 1976,101(21): 1314-1317.
3 Kaul V, Wani NA, Paljor YD. Primary carcinoma of gall bladder-a review of thirty six cases. Indian J Pathol Microbiol, 1989, 32(3): 146-151.
4 Orth K, gunz R, Staib L, et al. Gallbladder carcinoma as an incidental finding. Z Gastroenterol, 1991,29(12): 631-636.
5 Bosmans E, Onsea J, Verboven H. Gallbladder cancer as unexpected finding at cholecystectomy for benign disease. Acta Chit Belg. 1990, 90(5): 207-212.
6 Suzuki K, Kimura T, Ogawa H. Long-term prognosis of gallbladder cancer diagnosed after laparoscopic cholecystectomy. Surg Endosc, 2000, 14(8): 712-716.
7 Mori T, Souda S, Hashimoto j, et al. Unsuspected gallbladder cancer diagnosed by laparoscopic cholecystectomy: a clinicopathological study. Surg Today, 1997, 27(8): 710-713.
8 Kwon SY, Chang HJ. A clinicopathological study of unsuspected carcinoma of the gallbladder. J Korean Med Sci, 1997,12(6): 519-522.
9 Weinstein D, Herbert M, Bendet N, et al. Incidental finding of gallbladder carcinoma. Isr Med Assoc J, 2002,4(5): 334-336.
10 Bani-Hani KE, Yaghan RJ, Matalka Ⅱ, et al. Callbladder cancer in northern Jordan. J Gastroenterol Hepatol, 2003, 18(8) :954-959.
11 Frauenschuh D, Greim R, Kraas E. How to proceed in patients with carcinoma detected after laparoscopic cholecystectomy. Langenbecks Arch Surg, 2000, 385(8): 495-500.
12 Hohaus T, Hellmich G, Freitag M, Ludwig K. Gallbladder carcinoma—an unexpected finding after laparoscopic cholecystectomy. Zentralbl Chir. 1998,123 Suppl 2:80-83.
13 Glehen 0, Czyglik 0, Donsbeck AV,et al. Gallbladder cancers discovered fortuitously. Ann Chir, 2000, 125(2):137-143.
14 Antonakis P, Alexakis N, Mylonaki D, et al. Incidental finding of gallbladder carcinoma detected during or after laparoscopic cholecystectomy. Eur J Surg Oncol, 2003, 29(4):358-360.
15 Braghetto I, Bastias J, Csendes A, et al. Gallbladder carcinoma during laparoscopic cholecystectomy: is it associated with bad prognosis? Int Surg, 1999, 84(4):344-349.
16 Darabos N, Stare R. Gallbladder cancer: laparoscopic and classic cholecystectomy. Surg Endosc, 2004, 18 (1):144-147.
17 Romano F, Franciosi C, Caprotti R, et al. Gallbladder carcinoma and laparoscopic cholecystectomy. An emergent problem. Minerva Chir, 2000, 55 (12): 817-822.
18 Yeh CN, Jan YY, Chen MF. Management of unsuspected gallbladder carcinoma discovered during or following laparoscopic cholecystectomy. Am Surg, 2004, 70(3):256-258.
19 Gertsch P, Thomas P, Baer H, et al. Multiple tumors of the biliary tract. Am J Surg,1990, 159(4):386-388.
20 Chijiiwa K. Synchronous carcinoma of the gall-bladder in patients with bile duct carcinoma. Aust N Z J Surg, 1993, 63(9):690-692.
21 Washburn WK, Lewis WD, Jenkins RL. Liver transplantation with incidental gallbladder carcinoma in the recipient hepatectomy. HPB Surg, 1994, 8(2): 147-149.
22 Moser JJ, Baer HU, Glattli A, et al. Mirizzi syndrome-a contraindication for laparoscopic surgery. Helv Chir Acta, 1993, 59(4): 577-580.
23 Redaelli CA, Buchler MW, Schilling MK, et al. High coincidence of Mirizzi syndrome and gallbladder carcinoma. Surgery, 1997, 121(1): 58-63.
24 Ayhan A, Guney I, Saygan-Karamursel B, et al. Ovarian metastasis of primary biliary and gallbladder carcinomas. Eur J Gynaecol Oncol, 2001, 22(5): 377-378.
25 Schaeff B, Paolucci V, Thomopoulos J. Port site recurrences after laparoscopic surgery. A review. Dig Surg, 1998, 15: 124-134.
26 Zou SQ, Zhang L, Zen GZ. Clinical epidemiologic characteristics of 430 cases of gallbladder cancer. Chin Med J, 1998, 111(5): 391-393.
27 邹声泉，高戈，唐启彬，等．肝外胆管癌的细胞分子遗传学研究．第11届全国胆道外科学术会议论文汇编，2004，46-49．
28 Zou SQ, Zhang L. Relative risk factors analysis of 3922 cases of gallbladder cancer. China J Surg, 2000, 38(11): 805-808.
29 Napolitano L, Artese L, Innocenti P. Seeding from early stage gallbladder carcinoma after laparoscopic cholecystectomy. Ann Ital Chir, 2001, 72(6): 721-724.
30 Chorost MI, Huang IP, Webb H, et al. Incidental gallbladder carcinoma associated with a cholecystoduodenal fistula. Mil Med, 2002,167(10): 862-863.
31 Albores-Saavedra J, Shukla D, Carrick K, et al. In situ and invasive adenocarcinomas of the gallbladder extending into or arising from Rokitansky-Aschoff sinuses:a clinicopathologic study of 49 cases. Am J Surg Pathol, 2004, 28(5): 621-628.
32 Wade TP, Comitalo JB, Andrus CH, et al. Laparoscopic cancer surgery. Lessons from gallbladder cancer. Surg Endosc, 1994,8(6): 698-701.
33 Paolucci V, Schaeff B, Schneider M, et al. Tumor seeding following laparoscopy:international survey. World J Surg, 1999,23(10): 989-995.
34 Paotucci V, Neckell M, Gotze T. Unsuspected gallbladder carcinoma-the CAE-S/CAMIC registry. Zentralbl Chir, 2003,128(4): 309-312.
35 Suzuki K, Kimura T, Hashimoto H, et al. Port site recurrence of gallbladder cancer after laparoscopic surgery:two case reports of long-term survival. Surg haparosc Endosc Percutan Tech, 2000, 10(2): 86-88.
36 Cucinotta E, Lorenzini C, Lazzara S, et al. Intraperitoneal neoplastic dissemination of incidental gallbladder carcinoma after laparoscopic surgery. Tumori, 2003,89(4 Suppl): 34-39.
37 Z'graggen K, Birrer S, Maurer CA, et al. Incidence of port site recurrence after laparoscopic cbolecystectomy for preoperatively unsuspected gallbladder carcinoma. Surgery, 1998,124(5): 831-838.
38 石景森．胆道外科一个不可忽视的问题．第11届全国胆道外科学术会议论文汇编，2004，32-34．
39 Kim Hi, Roy T. Unexpected gallbladder cancer with cutaneous seeding after laparoscopic cholecystectomy. South Med J, 1994, 87(8): 817-820.
40 Sarli L, Costi R, Pietra N, et al. Incidental gallbladder cancer at laparoscopy: a review of two cases. Surg Laparosc Endosc Percutan Tech, 1999,9(6): 414-417.
41 Sailer M, Debus S, Fuchs KH, et al. Peritoneal seeding of gallbladder cancer after laparoscopic cholecystectomy. Surg Endosc, 1995, 9(12): 1298-1300.
42 Schauer RJ, Meyer G, Baretton G, et al. Prognostic factors and long-term results after surgery for gallbladder carcinoma: a retrospective study of 127 patients, hangenbecks Arch Surg, 2001,386(2): 110-117.
43 邹声泉．胆道肿瘤．见：邹声泉，龚建平主编．外科学—前沿与争论，第一版．北京：人民卫生出版社，2003：600-607．
2 Zou SQ, Zhang L, Zen GZ. Clinical epidemiologic characteristics of 430 cases of gallbladder cancer. Chin Med J, 1998,111(5): 391-393.
3 Zou SQ, Zhang L. Relative risk factors analysis of 3922 cases of gallbladder cancer. China J Surg, 2000, 38(11): 805-808.
4 黄志强．临床研究与新世纪胆道外科．第11届全国胆道外科学术会议论文汇编，2004，17-19．
5 邹声泉．胆道肿瘤．见：邹声泉，龚建平主编．外科学—前沿与争论，第一版．北京：人民卫生出版社，2003：600-607．
6 Vogelstein B, Kinz]er KW. Cancer genes and the pathways they control. Nat Med, 2004,10(8): 789-799.
7 邹声泉，高戈，唐启彬，等．肝外胆管癌的细胞分子遗传学研究．第11届全国胆道外科学术会议论文汇编，2004，46-49．
8 唐启彬，石雪涛，邹声泉，等．肝外胆管癌中染色体9p21区段抑癌基因簇表达异常的研究．肿瘤防治研究，2004，31(9)：523-525．
9 Zhao Y, Tan J, Zhuang L, et al. Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci USA. 2005 Nov 1; 102(44): 16090-16095.
10 Ohtani N, Yamakoshi K, Takahashi A, et al. The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression. J Med Invest, 2004 Aug; 51(3-4): 146-153.
1 Liu H, Dibling B, Spike B, et al. New roles for the RB tumor suppressor protein. Curr Opin Genet Dev. 2004 Feb; 14(1): 55-64.
2 Dasgupta P, Betts V, Rastogi S, et al. Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma protein: novel links between apoptotic signaling and cell cycle machinery. J Biol Chem. 2004 Sep 10; 279(37): 38762-9.
3 Mittnacht S. The retinoblastoma protein—from bench to bedside. Eur J Cell Biol. 2005 Mar; 84(2-3): 97-107.
4 Larsen CJ. pRB, p53, p16INK4a, senescence and malignant transformation. Bull Cancer. 2004 May; 91(5): 399-402.
5 Alland L, Muhle R, Hou HJ, et al. Role for N2CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature, 1997,387: 49.
6 Wang ZM, Yang H,Livingston DM. Endogenous E2F-1 promotes timely Go exit of resting mouse embryo fibroblasts. Proc Natl Acad Sci USA, 1998,95 (26): 15583-15586.
7 Simon B, Lubomierski N. Implication of the INK4a/ARF locus in gastroenteropancreatic neuroendocrine tumorigenesis. Ann N Y Acad Sci. 2004 Apr; 1014: 284-99.
1 Zhu L. Tumour suppressor retinoblastoma protein Rb: A transcriptional regulator. Eur J Cancer. 2005 Nov; 41(16):2415-27.
2 Niu Y, Li Y, Niu RF, et al. Correlation of E2F-1 and Rb expression with papillomatosis and ductal carcinoma in situ of the breast. Zhonghua Zhong Liu Za Zhi. 2004 May;26(5) :290-3.
3 Zhong M, Wang J, Zhang B, et al. Expression of pRb and E2F-1 and telomerase activity in ameloblastoma. Zhonghua Kou Qiang Yi Xue Za Zhi.2004 Sep; 39(5) :406-9.
4 Rogoff HA, Kowalik TF. Life, death and E2F:linking proliferation control and DNA damage signaling via E2F1. Cell Cycle. 2004 Jul; 3(7):845-6.
5 Pickering MT, Kowalik TF. Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene. 2005 Sep 26; [Epub ahead of print]
6 Jiang Y, Saavedra HI, Holloway MP, Leone G, Altura RA. Aberrant regulation of survivin by the RB/E2F family of proteins.J Biol Chem. 2004 Sep 24;279 (39): 40511-20.
7 Dasgupta P, Betts V, Rastogi S, et al.Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma protein: novel links between apoptotic signaling and cell cycle machinery. J Biol Chem. 2004 Sep 10;279(37):38762-9.
8 Gonzalo S, Blasco MA. Role of Rb family in the epigenetic definition of chromatin. Cell Cycle. 2005 Jun;4(6):752-5.
9 Luo RX, Postigo AA, Dean DC. Retinoblastoma interacts switch histone deacetylase to repress transcription. Cell, 1998, 92:496-473.
10 Magnaghi JL, Groisman R,Nagulbneva L, et al.Retinoblastoma protein repress transcription by recruiting a histone deacetyklase. Nature, 1998, 391(6667):601-605.
1 Dasgupta P, Betts V, Rastogi S, et al. Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma protein: novel links between apoptotic signaling and cell cycle machinery. J Biol Chem. 2004 Sep 10; 279(37): 38762-9.
2 Mittnacht S. The retinoblastoma protein—from bench to bedside. Eur J Cell Biol. 2005 Mar; 84(2-3): 97-107.
3 Larsen CJ. pRB, p53, p16INK4a, senescence and malignant transformation. Bull Cancer. 2004 May; 91(5): 399-402.
4 Alland L, Muhle R, Hou HJ, et al. Role for N2CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature, 1997, 387: 49.
5 Wang ZM, Yang H, Livingston DM. Endogenous E2F-1 promotes timely Go exit of resting mouse embryo fibroblasts. Proc Nat] Acad Sci USA, 1998,95 (26): 15583-15586.
6 Simon B, Lubomierski N. Implication of the INK4a/ARF locus in gastroenteropancreatic neuroendocrine tumorigenesis. Ann N Y Acad Sci. 2004 ipr; 1014: 284-99.
7 Claudio PP, DeLuca A, Howard CM, et al. Function alanalysis of pRb/p130 interaction with cyclins. Cancer Res, 1996, 56(9): 2003-2008.
8 He XS, Rong YH, Su Q, et al. Expression of p16 gene and Rb protein in gastric carcinoma and their clinicopathological significance. World J Gastroenterol. 2005 ipr 21; 11(15): 2218-2223.
1 Ikeguchi M, Oi K, Hirooka Y, Kaibara N. CD8+ lymphocyte infiltration and apoptosis in hepatocellular carcinoma. Eur J Suyg Oncol 2004; 30: 53-57
2 Dangles V, Validire P, Wertheimer M, Richon S, Bovin C, Zeliszewski D, Vallancien G, Bellet D. Impact of human bladder cancer cell architecture on autologous T-lymphocyte activation. Int J Cancer 2002; 98: 51-56
3 Zhao Y, Tan J, Zhuang L, et al. Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci USA. 2005 Nov 1; 102(44): 16090-5.
4 Abrahams VM, Kamsteeg M, Mot G. The Fas/Fas ligand system and cancer: immune privilege and apoptosis. Mol Biotechnol 2003; 25: 19-30
5 Griffith TS, Ferguson TA. The role of FasL-induced apoptosis in immune privilege. Imunol Today 1997; 18: 240-244
6 Humbert PO, Verona R, Trimarchi JM, et al. E2f3 is critical for normal cellular proliferation Genes Dev, 2000,14(6): 690-703.
7 Dimri GP, Itahana K, Acosta M, et al. Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14 (ARF) tumor suppressor. Mol Cell Biol, 2000, 20(1): 2732 285.
8 Frolov MY, Dyson NJ. Molecular mechanisms of E2F-dependent activation and pRB-mediated repression. J Cell Sci. 2004 May 1;117(Pt 11): 2173-81.
9 Pennaneach V, Barbier V, Regazzoni K, et al. Rb inhibits E2F-1-induced cell death in a LXCXE-dependent manner by active repression. J Biol Chem. 2004 May 28; 279(22): 23376-83.
1 Jenuwein T, Allis CD. Translating the histone code. Science, 2001, 203(5532): 1074-1080.
2 Geiman TM, Robertson KD. Chromatin remodeling, histone modifications, and DNA methylation-how does it all fit together? J Cell Biochem. 2002,87(2): 117-125.
3 Redner RL, Chen JD, Rush EA, et al. The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties. Blood. 2000,95(8): 2683-2690.
4 Richards EJ, Elgin SC. Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell. 2002,108(4): 489-450.
5 Luo RX, Postigo AA, Dean DC. Retinoblastoma interacts switch histone deacetylase to repress transcription. Cell, 1998, 92: 496-473.
6 Magnaghi JL, Groisman R, Nagulbneva h, et al. Retinoblastoma protein repress transcription by recruiting a histone deacetyklase. Nature, 1998, 391(6667): 601-605.
1 邹声泉，徐立宁．重视意外胆囊癌的诊治问题．中华外科杂志，2005，43(13)：833-5．
2 徐立宁，邹声泉．胆囊癌分子生物学研究现状及前景．实用肿瘤杂志，2005， 20(1)：7-9．
3 (美)萨比斯通，主编；王德炳，主译．克氏外科学，第十五版[M]．北京：人民卫生出版社，2000：15-32．
4 黄志强．胆道肿瘤．见：吴阶平，裘法祖主编．黄加驷外科学，第六版．北京：人民卫生出版社，2000：1290-3．
5 刘小方，邹声泉，裘法祖．肝炎病毒感染与肝门部胆管癌发病关系的探讨．中华外科杂志，2002，40(6)：420-422．
6 石景森．走出胆囊癌诊断的思维误区．临床外科杂志，2006，14(2)：68-69．
7 刘绍诰．胆囊癌．见：黄志强主编．当代胆道外科学，第一版．上海：上海科学技术文献出版社，1998：607-14．
8 Korenjak M, Brehm A. E2F-Rb complexes regulating transcription of genes important for differentiation and development. Curr Opin Genet Dev. 2005 Oct; 15(5): 520-7.
9 范祖森，敖世洲．肿瘤抑制基因RB与细胞周期调控研究新进展．生物化学与生物物理进展，1999，26(5)：429-433．
10 Zhu L. Tumour suppressor retinoblastoma protein Rb: A transcriptional regulator. Eur J Cancer. 2005 Nov; 41(16): 2415-27.
11 Niu Y, Li Y, Niu RF, et al. Correlation of E2F-I and Rb expression with papillomatosis and ductal carcinoma in situ of the breast. Zhonghua Zhong Liu Za Zhi. 2004 May; 26(5): 290-3.
12 Zhong M, Wang J, Zhang B, et al. Expression of pRb and E2F-1 and telomerase activity in ameloblastoma. Zhonghua Kou Qiang Yi Xue Za Zhi. 2004 Sep; 39(5): 406-9.
13 Rogoff HA, Kowalik TF. Life, death and E2F: linking proliferation control and DNA damage signaling via E2F1. Cell Cycle. 2004 Jul; 3(7): 845-6.
14 Pickering MT, Kowalik TF. Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene. 2005 Sep 26; [Epub ahead of print]
15 Jiang Y, Saavedra HI, Holloway MP, Leone G, Altura RA. Aberrant regulation of survivin by the RB/E2F family of proteins. J Biol Chem. 2004 Sep 24; 279(39): 40511-20.
16 Luo RX, Postigo AA, Dean DC. Retinoblastoma interacts switch histone deacety]ase to repress transcription. Cell, 1998, 92: 496-473.
17 Magnaghi JL, Groisman R, Nagulbneva L, et al. Retinoblastoma protein repress transcription by recruiting a histone deacetyklase. Nature, 1998, 391(6667): 601-605.
18 Pennaneach V, Barbier V, Regazzoni K, et al. Rb inhibits E2F-1-induced cell death in a LXCXE-dependent manner by active repression. J Biol Chem. 2004 May 28;279(22): 23376-83.
19 Zhao Y, Tan J, Zhuang L, et al. Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci USA. 2005 Nov 1; 102(44): 16090-5.
20 Narimatsu T, Tamori A, Koh N, et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology, 2004; 47(1): 26-31.
21 Wang ZW, Peng ZH, Li K, et al. Effect of p16 gene on carcinogenesis of hepatitis B virus related hepatocellular carcinoma. Zhonghua Zhong Liu Za Zhi, 2003 Jul; 25(4): 356-8.
22 刘小方，邹声泉．肝门部胆管癌中乙肝、丙肝病毒感染与p53异常表达的关系及意义．中华肝胆外科杂志，2003，9(3)：177-181．
23 Zou SO, Zhang L, Zen GZ. Clinical epidemiologic characteristics of 430 cases of gallbladder cancer. Chin Med J, 1998, 111(5):391-3.
24 Zou SQ, Zhang L. Relative risk factors analysis of 3922 cases of gallbladder cancer. China J Surg, 2000,38(11): 805-8.
25 邹声泉．胆道肿瘤．见：邹声泉，龚建平主编．外科学—前沿与争论，第一版．北京：人民卫生出版社，2003-600-7．
1 邹声泉，徐立宁．重视意外胆囊癌的诊治问题．中华外科杂志，2005，43(13)：833-5．
2 MARCIAL-ROJAS RA, MEDINA R. Unsuspected carcinoma of the gallbladder in acute and chronic cholecystitis. Ann Surg, 1961, 153: 289-98.
3 田华，陈力，刘桂杰，等．意外胆囊恶性肿瘤的外科治疗．中华外科杂志，2005，43(13)：836-8．
4 Zou SQ, Zhang L, Zen GZ. Clinical epidemiologic characteristics of 430 cases of gallbladder cancer. Chin Med J, 1998,111(5): 391-3.
5 Zou SQ, Zhang L. Relative risk factors analysis of 3922 cases of gallbladder cancer. China J Surg, 2000, 38(11): 805-8.
6 邹声泉．胆道肿瘤．见：邹声泉，龚建平主编．外科学—前沿与争论，第一版．北京：人民卫生出版社，2003：600-7．
1 Korenjak M, Brehm A. E2F-Rb complexes regulating transcription of genes important for differentiation and development. Curr Opin Genet Dev. 2005 Oct;15(5): 520-527.
2 Liu H, Dibling B, Spike B, et al. New roles for the RB tumor suppressor protein. Curr Opin Genet Dev. 2004 Feb; 14(1):55-64.
3 Sparkes MS, Sparkes MC, Willson MG, et al. Regional assignment of genes for human esterase D and retinoblastoma to chromosome band 13q14. Science, 1980, 208: 1042-1044.
4 Bookstein R, Lee H. Human retinobl astoma susceptibility gene: genomic organization and analysis of hetterozygous intragenic deletins mutants. Proc Natl Acad Sui USA, 1988, 85: 2210-2114.
5 Hong FD, Huang H, et al. Structure of the human retinoblastoma gene. Proc Natl Acad Sci USA, 1989, 86: 5502-5506.
6 黄倩，汪健，陶勇浩，等．正常人及视网膜母细胞瘤患者Rh基因启动于结构、功能和突变的研究．中华肿瘤，1998，20(3)：165-167．
7 Sowa Y, Shiioy, Fujita T, et al. Retinoblastoma binding factorl site in the core promotor region of the human retinoblastoma gene is activated byb GABP/ E4TF1.Cancer Res, 1997,57: 3145-3148.
8 Friend SH, Bernards R, Rogelj S, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature, 1986, 323: 643.
9 Lee WH, Bookatein R, tiong F, et al. Human retinoblastoma suscepitibility gene, cloning, identification and sequence. Science, 1987, 235: 1394.
10 Fung YKT, Murphree AL, T'Ang A, et al. Struetural evidence for the authenticity of the human recinoblastoma gene. Science, 1987,236: 1657.
11 Landschulze WH, Johnson PF, Mcknight SL. Tbe leucine zipper:a hypothetical structure common to a new class of DNA binding proteins. Science, 1988, 240: 1759-1763.
12 Ritety DJ, Liu CY, Lee WH, et al. Mutations of N-terminal regions renders the Retinoblastoma protein in sufficient functions in development and tumor suppression. Mol Col Biol,1997,17: 7342-7352.
13 Smith RC, Witts KN, Antelman M, et al. Adenovirial constructs econding phosphorylation competent full length and truncated forma of the human retinoblatoma protein inhibit myocyte proliferation and neointima formation. Circulation, 1997,96: 1899-1905.
14 Borges HL, Bird J, Wasson K, et al. Tumor promotion by caspase-resistant retinoblastoma protein. Proc Natl Acad Sci USA. 2005 Oct 25; 102(43): 15587-15592.
15 Dasgupta P,Betts V, Rastogi S, et al.Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma proteinrnovel links between apoptotic signaling and cell cycle machinery. J Biol Chem. 2004 Sep 10;279(37):38762-38769.
16 Ludlow JW, DeCaprio JA , Huang CM, et al. SV 40 large T antigen binds prefertially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell, 1989, 56:57-65.
17 Nishinaka T, Fu YH, Chen LI, et al.A unique cathepsin-like protase isolated from CV1 cells is involved in rapid degradation of retinoblastoma susceptibility gene product, RB, and transcription factor SPl.Biochim Biophys Acta, 1997, 1351:274-286.
18 Mittnacht S. The retinoblastoma protein—from bench to bedside.Eur J Cell Biol. 2005 Mar;84(2-3):97-107.
19 Larsen CJ. pRB, p53, pl6INK4a, senescence and malignant transformation. Bull Cancer.2004 May;91(5):399-402.
20 Angus SP, Mayhew CN, Solomon DA, et al. RB reversibly inhibits DNA replication via two temporally distinct mechanisms. Mol Cell Biol. 2004 Jun;24(12):5404-5420.
21 Mayhew CN, Perkin LM, Zhang X, Sage J, Jacks T, Knudsen ES. Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents. Oncogene. 2004 May 20;23(23):4107-4120.
22 Grana X, Reddy EP.Cell cycle control in mammlian cells:role of cyclins, cyclin dependent kinase (CDKs), growth suppressor and cyclin dependent kinase inhibitors (CDKs). J Oncogene, 1995, 11:21-26.
23 DeGregori J. The Rb network. J Cell Sci. 2004 Jul 15;117(Pt 16): 3411-3.
24 Zhu L. Tumour suppressor retinoblastoma protein Rb: A transcriptional regulator. Eur J Cancer. 2005 Nov;41(16):2415-2427.
25 Niu Y, Li Y, Niu RF, et al. Correlation of E2F-1 and Rb expression with papillomatosis and ductal carcinoma in situ of the breast. Zhonghua Zhong Liu Za Zhi. 2004 May; 26(5):290-293.
26 Zhong M, Wang J, Zhang B, et al. Expression of pRb and E2F-1 and telomerase activity in ameloblastoma. Zhonghua Kou Qiang Yi Xue Za Zhi. 2004 Sep; 39(5):406-409.
27 Rogoff HA, Kowalik TF. Life, death and E2F:linking proliferation control and DNA damage signaling via E2F1. Cell Cycle. 2004 Jul;3(7):845-846.
28 Pickering MT, Kowalik TF.Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation. Oncogene. 2005 Sep 26; [Epub ahead of print]
29 Jiang Y, Saavedra HI,Holloway MP, Leone G, Altura RA. Aberrant regulation of survivin by the RB/E2F family of proteins. J Biol Chem. 2004 Sep 24; 279(39):40511-40520.
30 Gonzalo S, Blasco MA. Role of Rb family in the epigenetic definition of chromatin. Cell Cycle. 2005 Jun;4(6):752-755.
31 Hernando E, Nahle Z, Juan G,et al. Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature. 2004 Aug 12; 430 (7001):797-802.
32 McCabe MT, Davis JN, Day ML. Regulation of DNA methyltransferase 1 by the pRb/E2F1 pathway. Cancer Res. 2005 May 1;65(9):3624-3632.
33 Zhao Y, Tan J, Zhuang L, et al.Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci USA. 2005 Nov 1;102(44):16090-16095.
34 Nagy L, Kao HY, Chakravarti D, et al. Nuclear recptor reprerssion mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell, 1997, 89:373.
35 Hassig CA, Fleischer TC, Billin AN et al. Histone deacetylase activity is requied for transcriptionsl repression by mSin3A. Cell, 1997,89:341.
36 Alland L, Muhle R, Hou HJ, et al. Role for N2CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature, 1997, 387:49.
37 Megee PC, Morgan BA, Smith MM. Histone H4 and the maintenance of genome integrity. Genes Dev,1995, 9:1716.
38 Yu X, Guo ZS, Marcu MG, et al. Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228. J Natl Cancer Inst, 2002, 94:504.
39 Liu L T, Chang HC, Chiang LC et al. Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion. Cancer Res, 2003, 63:3069.
40 Tsurutani J, Soda H, Oka M, et al. Antiproliferative effects of the histone deacetylase inhibitor FR901228 on small-cell lung cancer lines and drug-resistant sublines.Int J Cancer, 2003, 104:238.
41 Sakimura R, Tanaka K, Nakatani F, et al. Antitumor effects of histone deacetylase inhibitor on Ewing's family tumors. Int J Cancer. 2005 Sep 20;116(5):784-792.
42 Atadja P, Hsu M, Kwon P, et al. Molecular and cellular basis for the anti-proliferative effects of the HDAC inhibitor LAQ824. Novartis Found Symp. 2004;259:249-266.
43 Luo RX, Postigo AA, Dean DC. Retinoblastoma interacts switch histone deacetylase to repress transcription. Cell, 1998, 92:496-473.
44 Magnaghi JL, Groisman R, Nagulbneva L, et al. Retinoblastoma protein repress transcription by recruiting a histone deacetyklase. Nature, 1998, 391(6667):601-605.
45 Pennaneach V, Barbier V, Regazzoni K, et al. Rb inhibits E2F-1-induced cell death in a LXCXE-dependent manner by active repression. J Biol Chem, 2004 May 28; 279(22):23376-23383.
46 Jenuwein T, Allis CD. Translating the histone code. Science, 2001, 203(5532):1074-1080.
47 Geiman TM, Robertson KD. Chromatin remodeling, histone modifications, and DNA methylation-how does it all fit together? J Cell Biochem. 2002,87(2) : 117-125.
48 Redner RL, Chen JD, Rush EA, et al.The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties. Blood, 2000, 95(8):2683-2690.
49 Richards EJ, Elgin SC. Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell, 2002, 108(4): 489-450.
50 Wang ZM, Yang H, Livingston DM. Endogenous E2F-1 promotes timely G_0 exit of resting mouse embryo fibroblasts.Proc Natl Acad Sci USA, 1998,95 (26):15583-15586.
51 Slansky JE, Farnham PJ, Introduction to the E2F family:protein structure and gene regulation[J]. Curr ToD Microbiol Immunol, 1996,208:1.
52 Moberg KH, Logan TJ, Tyndall WA, et al. Three distinct elements within the murine c-myc promoter are required for transcription [J]. Oncogene, 1992, 7:411-412.
53 Leone G, DeGregori J, Yan Z, et al. E2F3 activity is regulated during the cell cycle and is required for the induction of S phase. Genes Dev, 1998, 12(14): 2120-2130.
54 Humbert PO, Verona R, Trimarchi JM, et al. E2f3 is critical for normal cellular proliferation. Genes Dev, 2000, 14(6):690-703.
55 Dimri GP, Itahana K, Acosta M, et al. Regulation of a senescence checkpoint response by the E2F1 transcription factor and p41(ARF) tumor suppressor. Mol Cell Biol, 2000, 20(1):273-285.
56 Frolov MV, Dyson NJ. Molecular mechanisms of E2F-dependent activation and pRB-mediated repression. J Cell Sci, 2004 May 1;117(Pt 10:2173-81.
57 Ginsberg D. E2F1 pathways to apoptosis. FEBS Lett,2002, 529(1): 122-125.
58 Krek W, Xu G, Livingston DM. Cyclin A-kinase regulation of E2F-1 DNA binding function underlies suppression of a S phase checkpoint. Cell, 1995, 83:1149-1158.
59 Rogoff HA, Pickering MT, Frame FM, et al. Apoptosis associated with deregulated E2F activity is dependent on E2F1 and Atm/Nbs1/Chk2. Mol Cell Biol, 2004 Apr; 24(7):2968-77.
60 Zacharatos P,Kot sinas A, Evangelou K. Distinct expression patterns of t he t ranscription factor E2F-1 in relation to tumour growth parameters in common human carcinomas. J Pat hol, 2004, 203 (3): 744-753.
61 Wyllie AH. E2F1 selects tumour cells for both life and death.J Pathol, 2002, 198(2):139-141.
62 Yang XH, Siadek TL. Overexpression of the E2F-1 transcription factor gene mediates cell transformation. Gene Expr, 1995,4:195-204.
63 Hunt KK, Deng J, Liu TJ, et al. Adenovirus mediated overexpression of the transcription factor E2F-1 induces apoptosis in human breast and ovarian2carcinoma cell lines and does not require P53. Cancer Res,1997, 57:4722-4726.
64 Balasubramanian S, Ahmad N, Mukhtar H. Upregulation of E2F transcription factors in chemically induced mouse skin tumors. Int J Oncol, 1999, 15(2) :387.
65 Pierce AM, Fisher SM, Conti CJ, et al. Deregulated expression of E2F1 induces hyperplasia and cooperates with ras in skin tumor development. Oncogene, 1998, 16(10):1267.
66 Dicker AJ, Popa C, Dahler AL, et al. E2F-1 induces proliferation-specific genes and suppresses squamous differenuiation-specific genes in human epidermal keratinocytes. Oncogene, 2000, 19(25) :2887.
67 Yamazaki K, Hasegawa M, Ohoka I, et al. Increased E2F-1 expression via tumour cell proliferation and decreased apoptosis are correlated with adverse prognosis in patients with squamous cell carcinoma of the oesophagus. J Clin Pathol, 2005 Sep;58(9):904-10.
68 Yamasaki L, Jacks T, Bronson, et al. Tumor induction and tissue atrophy in mice lacking E2F-1. Cell, 1996, 85:537-548.
69 Gorgoulis VG, Zacharatos P, Mariatos G, et al. Transcription factor E2F-1 act s as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas. J Pathol, 2002, 198(2): 142-156.
70 Eymin B, Gazzeri S, Brambilla C, et al. Distinct pattern of E2F1 expression in human lung tumours:E2F1 is upregulated in small cell lung carcinoma. Oncogene, 2001, 20(14):1678-1687.
71 DeMuth JP, Jackson CM, Weaver DA, et al. The gene expression index c-myc x E2F-1/p21 is highly predictive of malignant phenotype in human bronchial epit helial cells. Am J Respir Cell Mol Biol, 1998, 19(1): 18-24.
72 Yamasaki L. Balancing proliferation and apoptosis in vivo:the Goldilocks theory of E2FPDP action. Biochim Biophys Acta, 1999, 1423:M9-15.
73 Halaban R. Rb/E2F:a two-edged sword in the melanocytic system. Cancer Metastasis Rev, 2005 Jun24(2): 339-356.
74 O' Conner DJ, Lu X. Stress signals induce transcriptionally inactive E2F-1 independently of p53 and Rb. Oncogene, 2000,19(20): 2369-2376.
75 McCabe MT, Azih OJ, Day ML. pRb-Independent growth arrest and transcriptional regulation of E2F target genes. Neoplasia, 2005 Feb; 7(2): 141-51.
76 Thomas DW. Cellular senescence and cancer. J Pathology, 1999, 187: 100-111.
77 Claudio PP, DeLuca A, Howard CM, et al. Function alanalysis of pRb/p130 interaction with cyclins. Cancer Res, 1996,56(9): 2003-2008.
78 He XS, Rong YH, Su Q, et al. Expression of p16 gene and Rb protein in gastric carcinoma and their clinicopathological significance. World J Gastroenterol. 2005 Apr 21; 11(15): 2218-2223.
79 范祖森，敖世洲．肿瘤抑制基因RB与细胞周期调控研究新进展．生物化学与生物物理进展，1999，26(5)：429-433．
80 Ohlson LC, Koroxenidou L, Porsch-Hallstrom I. Mitoinhibitory effects of the tumor promoter 2-acetylaminofluorene in rat liver: loss of E2F-1 and E2F-3 expression and cdk 2 kinase activity in late G1. J Hepatol, 2004 Jun; 40(6): 957-62.
81 Serrano M, Hannon GJ, Beach D.A new regulatory motif in cell-cycle control causing specific inhibition of cyclinD/CDK4. Nature, 1993, 366(6456): 704-707.
82 Kamb A, Shattuck, Eeles R, et al. Ana]ysis of the P16 gene (CDKN2) usa candidate for the chromosome 9p melanoma susceptibility cocas. Science, 1994,264:436.
83 Sherr CJ. Mammalian G1 cyclin. Cell, 1993,73(6): 1059-1065.
84 Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 1994, 264(5157):436-440.
85 Simon B, Lubomierski N. Implication of the INK4a/ARF locus in gastroenteropancreatic neuroendocrine tumorigenesis. Ann N Y Acad Sci, 2004 Apr;1014:284-299.
86 Sharpless NE. INK4a/ARF: a multifunctional tumor suppressor locus. Mutat Res, 2005 Aug 25; 576(1-2):22-38.
87 Marx J . New tumor suppressor may rival p53. Science, 1994, 264(5157):344-345.
88 Schulze A , Zerf ass K, Spit kovsky D , et al . Activation of the E2F transcription factor by cyclin D1 is blocked by pl6INK4, the product of the putative tumor suppressor gene MST1. Ocogene, 1994, 9(12):3475-3482.
89 Nilsson K, Landberg G. Subcellular localization, modification and protein complex formation of the cdk-inhibitor p16 in Rb-functional and Rb-inactivated tumor cells. Int J Cancer, 2005 Sep 13; [Epub ahead of print]
90 Plath T, Detjen K, Welzel M, et al.A novel function for the tumor suppressor pl6INK4a: induction of anoikis via upregulation of the α 5β1 fibronectin receptor. J Cell Biol, 2000, 150(6) : 1467-1478.
91 Nobori T, Miura K, Wu DJ, et al. Deletions of the cycle dependent kinase-4 inhibitor gene in multiple human cancers. Nature, 1994, 368(6473):753-756.
92 Okamoto A, Demetrick DJ, Spillare EA, et al. Mutations and altered expression of pl6INK4 in human cancer. Proc Natl Acad Sci USA, 1994, 91(23): 11045-11049.
93 Xavier G. Cell cycle control in mammalian cells:role of cyclins, cyclin dependent kinases(CDKs), growth suppressor genes and cyclin dependent kinase inhibitors(CKIs). Oncogene, 1995, 11:211-219.
94 Liggett WH Jr, Sidransky D. Role of the pl6 tumor suppressor gene in cancer. J Clin Oncol, 1998, 16(3):1197-1206.
95 Shapiro GI, Edwards CD, Kobzik L, et al. Reciprocal Rb in activation and p16INK4 expressionin primary lung cancers and cell lines. Cancer Res, 1995, 55:505-509.
96 Ohtani N, Yamakoshi K,Takahashi A, et al.The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression. J Med Invest,2004 Aug;51(3-4):146-153.
97 Serrano M, Grogory J, Hannon, et al. A new regulatory motif in cellcycle control causing specific inhibition of cycling D/cdk4. Nature, 1993, 366:704.
98 Noborl T,Kaoru Mlura, David J, et al.Deletions of the cycling dependent kinase 4 inhibitor hence in multiple human cancers. Nature, 1994,368:753.
99 Cairns P, Polascik TJ, Eby Y, et al. Frequency of homozygous deletion at P16 CKDN2 in primary tumors. Nature Genetics,1995, 11:210-212.
100 Volgareva GM, Zavalishina LE, Andreeva IuIu, et al. Expression of pl6INK4a in various cancer cells. Arkh Patol, 2004 Sep-Oct ;66(5) :3-5.
101 Nilsson K, Svensson S, Landberg G. Retinoblastoma protein function and p16INK4a expression in actinic keratosis, squamous cell carcinoma in situ and invasive squamous cell carcinoma of the skin and links between pl6INK4a expression and infiltrative behavior. Mod Pathol, 2004 Dec;17(12):1464-1474.
1 (美)萨比斯通，主编；王德炳，主译．克氏外科学，第十五版[M]．北京：人民卫生出版社，2000：15-32．
2 石景森．我国胆囊癌的发病情况及外科处理[J]．肝胆胰外科杂志，1999， 11(1)：51-52．
3 Merimsky O, Issakov J, Schwartz I, et al. Lack of ErbB-2 oncogene product overexpression in soft tissue sarcomas[J]. Acta Oncol, 2002, 41(4): 366-368.
4 Kamel D, Paakko P, Nuorva K, et al. p53 and c-erbB-2 protein expression in adenocarcinomas and epithelial dysplasias of the gallbladder[J]. J Pathol, 1993, 170(1): 67-72.
5 Chow NK, Huang SM, Chang SH. Significance of C-erbB-2 expression normal and neoplastic epithelium of biliary tract[J]. Anticancer Res, 1995, 15: 1055.
6 Akisik E, Bavbek S, Dalay N. CD44 variant exons in leukemia and lymphoma[J]. Pathol Oncol Res, 2002, 8(1): 36-40.
7 Gunthert U, Hofmann M, Rudy W, et al. A new variant of glycoprotein CD44confersmetastasis potential to rat carcinomacells[J]. Cell, 1991, 65: 13-24.
8 谷化平，尚培中，刘艳茹．原发性胆囊癌CD44v6和Bcl-2的表达及其与临床病理的联系[J]．中国普外基础与临床杂志，2003，10(1)：46-48．
9 Steeg PS, Bevilacqua G, Kopper L, et al. Evidence for a novel gene associated witha low tumor metastatic potential[J]. J Natl Cancer Inst, 1988, 80(17): 204-208.
10 吴晓华，沈忠英，汤雪明．肿瘤转移抑制基因rim23研究进展[J]．生命科学，1996，8(3)：18-21．
11 Hennessy C, Henry JA, May FE, et al. Expression of the antimetastatic gene nm23 in human breast cancer: an association with good prognosis[J]. J Natl Cancer Inst, 1991, 83(4): 281-284.
12 Galigo MA, Cipollini G, Berti A, et al. rim23 gene expression in human advanced phase of tumor progression[J]. Int Cancer, 1997, 74(1): 102-107.
13 Huang G, Song Y, He G. mRNA expression and mutation of MTA1 and nm23H1 genes in ovarian carcinoma in relation to lymph node metastasis[J]. Zhonghua ZhongLiu ZaZhi., 2001, 23(1): 31-34.
14 Galani E, Sgouros J, Petropoulou C, et al. Correlation of MDR-1, nm23-H1 and H Sema E gene expression with histopathotogical findings and clinical outcome in ovarian and breast cancer patients[J]. Anticancer Res, 2002, 22(4): 2275-2280.
15 Forte A, D'Urso A, Gallinaro LS, et al. NM23 expression as prognostic factor in colorectal carcinoma[J]. G Chir, 2002, 23(3): 61-63.
16 Belev B, Aleric I, Vrbanec D, et al. Nm23 gene product expression in invasive breast cancer-immunohistochemical analysis and clinicopathological correlation[J]. Acta Oncol, 2002, 41(4): 355-361.
17 谷化平，尚培中，刘艳茹．nm23-H1和E-Cadherin基因蛋白在胆囊癌组织中的表达[J]．中国现代普通外科进展，2002，5(1)：29-31．
18 邹声泉．胆囊癌的分子生物学研究现状和进展[J]．世界华人消化杂志，1999，7(11)：926-927．
19 Imam M, Hoshi T, Ogawa K. K-ras codon 12 mutations in biliary tract tumors detected by polymerase chain reaction denaturing gradient gel ectrophrosis[J]. Cancer, 1994, 73: 2727.
20 Ajiki T, Fujimor H, Onoyyama M. K-ras gene mutation in gallbladder carcinoma and dysplasia[J]. Gut, 1996, 38: 426-429.
21 Matsubara T, Sakurai Y, Sasyama Y. K-ras point mutation in cancerous and noncancerous biliary epithelium in patient with pancreaticobiliary maljunction[J]. Cancer, 1996, 77: 1752-1757.
22 卿德科，夏亮芳，庞家芳．原发性胆囊癌FaS p21、PCNA、AgNORs的对比研究．中国肿瘤临床，1999，26(1)：59-60．
23 Hahn SA, Schutte M, Hoque AT, et al. DPC4 a candidate tumor suppressor gene at human chromosome 18q21.1[J]. Science, 1996, 19(271): 350-353.
24 Riggins GJ, Kinzler KW, Vogelstein B, et al. Frequency of Smad gene mutations in human cancer[J]. Cancer Res, 1997, 57: 2578-2580.
25 Lagna G, Hata A, Hemmati BA, et al. Partnership between DPC4 and Smad proteins in TGFβ Signaling pathways[J]. Nature, 1996, 383: 832-836.
26 Gustin A, Pederson L, Miller R, et al. Application of molecular biology studies to gene therapy treatment strategies[J]. World J Surg, 2002, 26(7): 854-860.
27 Massague J. TGFβ signaling: receptors, transducers and Mad proteins[J]. Cell, 1996, 85: 947-950.
28 Schutte M. DPC4 gene alterations in human cancer, and their functional implications[J]. Ann Oncol, 1999, 10(4): 56-59.
29 陈军，胡志前，蒋树娟，等．原发性胆囊癌抑癌基因DPC4蛋白表达及其意义[J]．肝胆胰外科杂志，2002，14(1)：52-53．
30 Santoni RE, Duro D, Farkas T, et al. E2Factivity is essential for survival of Myc-overexpressing human cancer cells[J]. Oncogene, 2002, 21(42): 6498-6509.
31 Rim ttS, Shin JY, Ahn DK, et al. Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large W antigen[J]. Exp Mol Med, 2001, 33(4): 293-298.
32 梁刚，沈陶然，徐小宏，等．早期胆囊癌癌基因的表达[J]．临床与实验病理学杂志， 1997，13(4)：333-335．
33 龚非力．医学免疫学，第一版[M]．北京：科学出版社，2000：261．
34 范毓东．Bcl-2与胆管细胞癌[J]．国外医学外科学分册，1999，26(3)：159-161．
35 Tsujimoto Y, Finger LR, Yunis J, et al. Cloning of the chromosome breakpoint neoplastic B cells with the t(14;18) chromosome translocation[J]. Science, 1984, 226(30): 1097-1099.
36 谷化平，尚培中，周翠玲．胆囊癌组织CD15和Bcl-2的表达及相关性研究[J]．临床肝胆病杂志，2001，17(1)：57-58．
37 Itoh N, Tsujimoto Y, Nagata S. Effect of bcl-2 on fas antigen-mediated cell death[J]. J Immunol, 1993, 151(2): 621-627.
38 Mikami T, Yanagisawa N, Baba H, et al. Association of bcl-2 protein expression with gallbladder carcinoma differentiation and progression and its relation toapoptosis[J]. Cancer, 1999, 85(2): 318-325.
39 徐立宁，丁志强．胆囊癌组织Bcl-2蛋白和Fas／FasL蛋白的表达及相关性研究[J]．中国现代医学，2003，13(4)：28-30．
40 Gao Y, FergusonDO, Xie W, et al. Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development[J]. Nature, 2000, 404(6780): 897-900.
41 Difilippantonio M, Petersen S, Chen HT, et al. Evidence for replicative repair of DNA double-strand breaks leading to oncogenie translocation and gene amplification[J]. J Exp Med, 2002, 196(4): 469-80.
42 Matsubara T. K-ras point mutations in cancerous and noncancerous biliary epithelium inpatient with pancreaticobiliary maljunction[J]. Cancer, 1996, 77: 1752.
43 Sarpa A. Neoplasma of the ampwla of vater K-ras and p53 mutations[J], am J Pathol, 1993, 142: 1163.
44 Serrano M, Harmon GJ, Beach DA. A new regulatory motifin cell-cycle control causing specific inhibition of cyclin D/CDK4[J]. Nutune (lond), 1993, 366(6456): 704-707.
45 Kamb A, Gruis NA, Weaver FJ, et al. A cell cycle regulator potentially involved in genesis of many tumor types[J]. Science, 1994, 264(5157): 436-440.
46 Ku JL, Yoon KA, Kim IJ, et al. Establishment and characterisation of six human biliary tract cancer cell lines[J]. Br J Cancer, 2002, 87(2): 187-193.
47 马红兵，狄政莉，王作仁，等．抑癌基因p16、Rb的表达与胆囊癌预后的关系[J]．西安医科大学学报，2001，22(4)-343-345．
1 MARCIAL-ROJAS RA, MEDINA R. Unsuspected carcinoma of the gallbladder in acute and chronic cholecystitis. Ann Surg, 1961, 153: 289-298.
2 Andersson A, Bergdahl L, Zeuchner E. Carcinoma as an unexpected histologic diagnosis in gallbladder disease, gentralbl Chir, 1976,101(21): 1314-1317.
3 Kaul V, Wani NA, Paljor YD. Primary carcinoma of gall bladder-a review of thirty six cases. Indian J Pathol Microbiol, 1989, 32(3): 146-151.
4 Orth K, gunz R, Staib L, et al. Gallbladder carcinoma as an incidental finding. Z Gastroenterol, 1991,29(12): 631-636.
5 Bosmans E, Onsea J, Verboven H. Gallbladder cancer as unexpected finding at cholecystectomy for benign disease. Acta Chit Belg. 1990, 90(5): 207-212.
6 Suzuki K, Kimura T, Ogawa H. Long-term prognosis of gallbladder cancer diagnosed after laparoscopic cholecystectomy. Surg Endosc, 2000, 14(8): 712-716.
7 Mori T, Souda S, Hashimoto j, et al. Unsuspected gallbladder cancer diagnosed by laparoscopic cholecystectomy: a clinicopathological study. Surg Today, 1997, 27(8): 710-713.
8 Kwon SY, Chang HJ. A clinicopathological study of unsuspected carcinoma of the gallbladder. J Korean Med Sci, 1997,12(6): 519-522.
9 Weinstein D, Herbert M, Bendet N, et al. Incidental finding of gallbladder carcinoma. Isr Med Assoc J, 2002,4(5): 334-336.
10 Bani-Hani KE, Yaghan RJ, Matalka Ⅱ, et al. Callbladder cancer in northern Jordan. J Gastroenterol Hepatol, 2003, 18(8) :954-959.
11 Frauenschuh D, Greim R, Kraas E. How to proceed in patients with carcinoma detected after laparoscopic cholecystectomy. Langenbecks Arch Surg, 2000, 385(8): 495-500.
12 Hohaus T, Hellmich G, Freitag M, Ludwig K. Gallbladder carcinoma—an unexpected finding after laparoscopic cholecystectomy. Zentralbl Chir. 1998,123 Suppl 2:80-83.
13 Glehen 0, Czyglik 0, Donsbeck AV,et al. Gallbladder cancers discovered fortuitously. Ann Chir, 2000, 125(2):137-143.
14 Antonakis P, Alexakis N, Mylonaki D, et al. Incidental finding of gallbladder carcinoma detected during or after laparoscopic cholecystectomy. Eur J Surg Oncol, 2003, 29(4):358-360.
15 Braghetto I, Bastias J, Csendes A, et al. Gallbladder carcinoma during laparoscopic cholecystectomy: is it associated with bad prognosis? Int Surg, 1999, 84(4):344-349.
16 Darabos N, Stare R. Gallbladder cancer: laparoscopic and classic cholecystectomy. Surg Endosc, 2004, 18 (1):144-147.
17 Romano F, Franciosi C, Caprotti R, et al. Gallbladder carcinoma and laparoscopic cholecystectomy. An emergent problem. Minerva Chir, 2000, 55 (12): 817-822.
18 Yeh CN, Jan YY, Chen MF. Management of unsuspected gallbladder carcinoma discovered during or following laparoscopic cholecystectomy. Am Surg, 2004, 70(3):256-258.
19 Gertsch P, Thomas P, Baer H, et al. Multiple tumors of the biliary tract. Am J Surg,1990, 159(4):386-388.
20 Chijiiwa K. Synchronous carcinoma of the gall-bladder in patients with bile duct carcinoma. Aust N Z J Surg, 1993, 63(9):690-692.
21 Washburn WK, Lewis WD, Jenkins RL. Liver transplantation with incidental gallbladder carcinoma in the recipient hepatectomy. HPB Surg, 1994, 8(2): 147-149.
22 Moser JJ, Baer HU, Glattli A, et al. Mirizzi syndrome-a contraindication for laparoscopic surgery. Helv Chir Acta, 1993, 59(4): 577-580.
23 Redaelli CA, Buchler MW, Schilling MK, et al. High coincidence of Mirizzi syndrome and gallbladder carcinoma. Surgery, 1997, 121(1): 58-63.
24 Ayhan A, Guney I, Saygan-Karamursel B, et al. Ovarian metastasis of primary biliary and gallbladder carcinomas. Eur J Gynaecol Oncol, 2001, 22(5): 377-378.
25 Schaeff B, Paolucci V, Thomopoulos J. Port site recurrences after laparoscopic surgery. A review. Dig Surg, 1998, 15: 124-134.
26 Zou SQ, Zhang L, Zen GZ. Clinical epidemiologic characteristics of 430 cases of gallbladder cancer. Chin Med J, 1998, 111(5): 391-393.
27 邹声泉，高戈，唐启彬，等．肝外胆管癌的细胞分子遗传学研究．第11届全国胆道外科学术会议论文汇编，2004，46-49．
28 Zou SQ, Zhang L. Relative risk factors analysis of 3922 cases of gallbladder cancer. China J Surg, 2000, 38(11): 805-808.
29 Napolitano L, Artese L, Innocenti P. Seeding from early stage gallbladder carcinoma after laparoscopic cholecystectomy. Ann Ital Chir, 2001, 72(6): 721-724.
30 Chorost MI, Huang IP, Webb H, et al. Incidental gallbladder carcinoma associated with a cholecystoduodenal fistula. Mil Med, 2002,167(10): 862-863.
31 Albores-Saavedra J, Shukla D, Carrick K, et al. In situ and invasive adenocarcinomas of the gallbladder extending into or arising from Rokitansky-Aschoff sinuses:a clinicopathologic study of 49 cases. Am J Surg Pathol, 2004, 28(5): 621-628.
32 Wade TP, Comitalo JB, Andrus CH, et al. Laparoscopic cancer surgery. Lessons from gallbladder cancer. Surg Endosc, 1994,8(6): 698-701.
33 Paolucci V, Schaeff B, Schneider M, et al. Tumor seeding following laparoscopy:international survey. World J Surg, 1999,23(10): 989-995.
34 Paotucci V, Neckell M, Gotze T. Unsuspected gallbladder carcinoma-the CAE-S/CAMIC registry. Zentralbl Chir, 2003,128(4): 309-312.
35 Suzuki K, Kimura T, Hashimoto H, et al. Port site recurrence of gallbladder cancer after laparoscopic surgery:two case reports of long-term survival. Surg haparosc Endosc Percutan Tech, 2000, 10(2): 86-88.
36 Cucinotta E, Lorenzini C, Lazzara S, et al. Intraperitoneal neoplastic dissemination of incidental gallbladder carcinoma after laparoscopic surgery. Tumori, 2003,89(4 Suppl): 34-39.
37 Z'graggen K, Birrer S, Maurer CA, et al. Incidence of port site recurrence after laparoscopic cbolecystectomy for preoperatively unsuspected gallbladder carcinoma. Surgery, 1998,124(5): 831-838.
38 石景森．胆道外科一个不可忽视的问题．第11届全国胆道外科学术会议论文汇编，2004，32-34．
39 Kim Hi, Roy T. Unexpected gallbladder cancer with cutaneous seeding after laparoscopic cholecystectomy. South Med J, 1994, 87(8): 817-820.
40 Sarli L, Costi R, Pietra N, et al. Incidental gallbladder cancer at laparoscopy: a review of two cases. Surg Laparosc Endosc Percutan Tech, 1999,9(6): 414-417.
41 Sailer M, Debus S, Fuchs KH, et al. Peritoneal seeding of gallbladder cancer after laparoscopic cholecystectomy. Surg Endosc, 1995, 9(12): 1298-1300.
42 Schauer RJ, Meyer G, Baretton G, et al. Prognostic factors and long-term results after surgery for gallbladder carcinoma: a retrospective study of 127 patients, hangenbecks Arch Surg, 2001,386(2): 110-117.
43 邹声泉．胆道肿瘤．见：邹声泉，龚建平主编．外科学—前沿与争论，第一版．北京：人民卫生出版社，2003：600-607．