顺铂诱导肿瘤细胞衰老相关新基因的筛选及功能鉴定
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摘要
研究背景与目的
迄今为止，许多类型的人类和动物细胞都能够体外培养，然而大部分培养细胞的增殖能力都有一定限度，当细胞增殖到一定阶段后就会进入不分裂的阶段，也就意味着进入了衰老(senescence)。自Hayflick上世纪60年代从人类纤维原细胞中发现了这种现象后，人们逐渐发现人类其它各类细胞和其它物种的细胞也存在着衰老。当人体受到各种有害因素刺激时，局部组织的细胞会产生异常增生从而形成肿瘤。通过对细胞衰老的不断研究，人们逐渐对衰老与肿瘤关系有了一种新的认识，认为细胞衰老的抗肿瘤作用是机体对抗肿瘤的一种重要机制。有很多体外试验表明，细胞受癌基因刺激后是通过发生衰老这一途径来抑制肿瘤形成的，更多的体内研究也证实了这一作用。然而，由于细胞衰老机制障碍，还是有很多正在发生恶性转化的细胞绕过衰老抑制而最终成为恶性肿瘤细胞。现有体内和体外的研究证实一些具有DNA损伤作用的化疗药物如顺铂、喜树碱、依托铂苷等，能够引起肿瘤细胞的加速性衰老(acceleratedsenescence)。也有一些研究证实ATM基因相关的DNA损伤修复通路中的衰老相关调控基因参与了化疗诱导的加速性衰老的发生，但是其具体机制仍不是十分清楚。因此本研究采用顺铂这一临床上最常用的化疗药物分别诱导NG108-15，Hela，A2780三株不同组织类型的肿瘤细胞系，试图阐明相关衰老调控基因在其中所发挥的作用。
方法
一、应用不同浓度梯度顺铂作用肿瘤细胞，选择能够达到最高衰老率而无明显凋亡发生的浓度作为实验浓度。
二、运用细胞免疫荧光检测肿瘤细胞衰老过程中HP1-γ蛋白(衰老相关异染色质灶)的表达变化；应用SA-βGal(衰老相关β-半乳糖苷酶)染色法检测顺铂诱导肿瘤细胞衰老情况。
三、采用MTT法和CFSE染色法检测顺铂诱导肿瘤细胞衰老过程中的细胞增殖情况；
四、选用咖啡因抑制ATM基因；PI单染流式细胞仪检测细胞周期变化；Western blot检测P53、P21、CDC2等衰老相关调控基因表达变化情况；
结果
在一定小剂量顺铂诱导下，NG108-15、Hela、A2780细胞均呈现出衰老表型。细胞变大变扁平，胞浆空泡增多，SA-βGal染色呈阳性着色，HP1-γ蛋白表达增多并呈灶状聚集分布在细胞核。衰老细胞增殖几乎停滞，细胞周期主要阻滞在G2／M期。ATM抑制剂咖啡因可以抵抗顺铂诱导的衰老发生。P53、P21、CDC2基因在顺铂诱导衰老过程中表达量和活性明显发生改变。
结论
一定小剂量的顺铂可以诱导NG108-15、Hela、A2780细胞发生加速性衰老。DNA损伤修复通路上的ATM、P53、P21、CDC2等基因参与了顺铂诱导的肿瘤细胞衰老过程；抑制ATM基因表达后可以逆转顺铂诱导的肿瘤细胞衰老。
第二部分利用二维电泳技术筛选新的顺铂诱导衰老相关基因
研究背景与目的
二维电泳技术是迄今为止蛋白质组学研究中使用最广泛的一种蛋白质分离技术，通过在第一向的等电聚焦，不同的蛋白质依照其等电点的大小聚集，继而在第二向的SDS-PAGE凝胶电泳将相同等电点的蛋白质依照其分子量大小的不同而进行分离。根据以上原理，二维电泳能够一次性将大量的不同等电点、不同分子量的蛋白质进行分离。鉴于二维电泳技术的高通量的优势，已有相当多的研究采用该技术研究肿瘤发生、发展过程中的相关蛋白质。细胞衰老指细胞从具有活性增殖的状态通过各种因素的影响而成为不可逆转的生长停滞状态。肿瘤细胞通常被认为存在衰老通路障碍。然而最近的研究证实无论是在体内或是体外实验中，一些DNA损伤因子如顺铂等化疗药物可以诱导肿瘤细胞发生衰老。然而究竟有哪些基因参与其中，这些基因与衰老的关系如何等均不是十分清楚。因此本实验选取二维电泳技术作为主要研究手段，系统筛选分析顺铂诱导肿瘤细胞衰老过程中相关差异表达蛋白，以期找到新的衰老相关调控基因。
方法
一、以一定剂量顺铂诱导NG108-15细胞衰老，提取细胞总蛋白；
二、采用二维电泳法筛选NG108-15细胞衰老前后表达差异蛋白。实验重复3次，选取表达差异在1.5倍以上的蛋白点进行后续分析。
三、MALDI-TOF-MS质谱分析鉴定选取蛋白质
四、Western blot、细胞免疫荧光验证二维电泳筛选出的差异蛋白的表达情况；构建裸鼠皮下瘤模型，瘤根原位注射顺铂。冰冻切片免疫组化法检测GRP78蛋白表达变化情况，SA-β-Gal染色法检测瘤体细胞衰老情况；
结果
对照SDS-PAGE胶中共有517±25个点被检测出，衰老NG108-15细胞对应的胶中共有474±21点被检测出。选取二维电泳胶中NG108-15细胞衰老前后5个表达差异明显的蛋白点进行后续分析。MALDI-TOF MS质谱分析共检测出5个差异蛋白。Western blot验证了Vimentin和GRP78在衰老前后的表达变化与二维电泳结果一致。Western blot、细胞免疫荧光和荷瘤裸鼠模型均证实GRP78在顺铂诱导NG108-15细胞发生衰老后表达明显降低。
结论
作为高通量筛选差异蛋白的有效技术手段，二维电泳技术可被成功用于筛选新的顺铂诱导肿瘤细胞衰老相关调控基因。GRP78蛋白在顺铂诱导NG108-15细胞衰老的过程中表达明显降低，可能与顺铂诱导的衰老存在某种联系。
第三部分GRP78基因在顺铂诱导肿瘤细胞衰老过程中的功能鉴定
研究背景与目的
葡萄糖调节蛋白78(glucose regulated p rotein，GRP78)，通常也被称为免疫球蛋白重链结合蛋白(the immu-noglobulin heavy chain binding p rotein，Bip)，与热休克蛋白70家族有非常高的同源性，一般认为它是热休克蛋白70家族中的一种蛋白质。GRP78参与了细胞的许多生理和病理过程，其生物学功能很复杂。它既是一种分子伴侣在蛋白质的折叠和转运中起着重要的作用，又是定位于内质网上的一种应激蛋白，在低糖、低氧、低Ca2 +等应激状态下其表达明显升高，进而保护细胞免受损伤。近年来研究表明GRP78在某些肿瘤细胞中表达较高，能够赋予肿瘤细胞抗凋亡的特性，并能够影响肿瘤细胞的抗原性。最近研究认为当星型胶质细胞受到重金属刺激的时候会高表达GRP78，从而达到保护神经元细胞免受损害的效果。肿瘤多发生于年龄较大的人群，与年龄存在着一定的相关性。1961年Hayflick在对体外培养细胞研究中发现细胞的增值并不是无限的，也有一定的限制性，他把这种现象叫做细胞衰老。迄今为止对细胞衰老机制方面的研究通常包括对细胞周期、自由基及DNA损伤与衰老的关系、衰老的端粒学说、线粒体与衰老、衰老相关基因和长寿基因、细胞信号转导等方面的研究。然而有关GRP78与衰老之间关系的研究还未见文献报道。本实验室前期通过二维电泳等技术证明GRP78基因在顺铂诱导NG108-15细胞衰老过程中表达明显降低，其可能是新的顺铂诱导肿瘤细胞衰老的相关调控基因。因此本研究拟对GRP78基因在顺铂诱导肿瘤细胞衰老中所扮演的角色进行深入探讨，以期阐明相关调控机制，为临床化疗提供新的治疗靶点和有效方案。
方法
一、Western blot分别检测GRP78在顺铂诱导NG108-15、Hela、A2780细胞衰老过程中的表达变化情况；
二、SiGRP78通过脂质体法转染肿瘤细胞，Western blot验证转染效果并检测相关衰老调控基因变化情况。SA-β-Gal染色检测封闭GRP78后肿瘤细胞对顺铂诱导衰老的敏感性变化情况；
三、应用GRP78诱导剂2DG和A23187上调GRP78在不同肿瘤细胞系的表达，SA-β-Gal染色检测上调GRP78蛋白后肿瘤细胞对顺铂诱导衰老的敏感性变化情况；Western blot检测上调GRP78后顺铂诱导衰老过程中P53、P21等衰老相关调控基因表达变化情况；
四、应用激光共聚焦显微镜检测细胞胞浆内钙离子浓度相对变化情况；
五、免疫组化法检测GRP78蛋白及HP1-γ蛋白在宫颈癌和卵巢癌中的表达情况。
结果
一、Western blot结果显示GRP78在NG108-15、Hela、A2780细胞顺铂诱导衰老过程中表达降低。Western blot结果显示CASPASE7在衰老过程中始终以无活性形式存在，提示在顺铂诱导肿瘤细胞衰老过程中无内质网凋亡途径激活。
二、在NG108-15、Hela、A2780细胞中利用GRP78SiRNA封闭GRP78表达后，P53表达均明显升高；
三、在NG108-15、Hela和A2780细胞中利用SiRNA下调GRP78表达后细胞对顺铂诱导的衰老敏感性略有增加。利用SiRNA下调卵巢癌顺铂耐药细胞C13K的GRP78表达后衰老相关β半乳糖苷酶染色阳性率明显升高。
四、2DG诱导GRP78表达上调后NG108-15细胞对顺铂诱导衰老的敏感性明显降低，接着SiRNA下调GRP78表达后敏感性恢复。而在Hela和A2780细胞中则无明显改变。在Hela和A2780细胞中A23187诱导GRP78表达上调后细胞对顺铂诱导衰老的敏感性显著降低，SiRNA下调GRP78表达后敏感性恢复。
五、Western blot结果显示上调GRP78表达后顺铂诱导肿瘤细胞衰老过程中P53表达升高趋势明显被抑制。
六、胞浆内钙离子荧光强度检测显示诱导衰老剂量的顺铂作用NG108-15细胞24小时后胞浆内钙离子浓度升高，而上调GRP78后顺铂作用24小时胞浆内钙离子浓度无明显升高。诱导剂诱导GRP78高表达后接着用SiRNA下调GRP78表达，则顺铂作用24小时时胞浆内钙离子浓度显著升高。
七、免疫组化结果显示GRP78在宫颈癌的正常组织以及癌前病变和原位癌中呈高表达，而在癌组织中的表达较低。GRP78在卵巢正常和良性组织中表达较低，而在卵巢癌中呈高表达。在各种肿瘤组织GRP78表达与HP1-γ表达具有高度相关性。(P＜0.05)
结论
一、GRP78作为新发现的化疗介导的衰老相关调控基因，与P53、P21、CDC2等DNA损伤修复基因具有一定的相互拮抗或促进作用；下调GRP78的表达能够显著提高P53基因的表达水平，而上调其表达则能够显著降低肿瘤细胞对顺铂诱导衰老的敏感性。提示GRP78在顺铂诱导肿瘤衰老过程中主要扮演抵抗衰老的角色。
二、GRP78可能通过影响DNA损伤修复通路基因的表达及胞浆内钙离子浓度的改变从而影响肿瘤细胞对顺铂诱导衰老的敏感性。在宫颈癌癌前病变中GRP78的表达明显高于正常组织和癌组织，且与HP1-γ蛋白的表达具有很强的关联性，提示其可能参与了正常细胞向癌细胞恶性转化的过程。在卵巢癌中GRP78呈高表达，提示其可能通过抗衰老途径影响卵巢癌化疗效果。
第四部分应用激光扫描细胞仪分析GRP78蛋白和HP1-γ蛋白在食管癌各级病变中的表达情况及两者表达相关性的研究
研究背景和目的
1961年Hayflick在对体外培养细胞研究中发现细胞有增殖极限的现象，并称之为细胞衰老。以往的观点认为，肿瘤细胞具有无限制增殖的永生化能力，因此被认为存在一定的衰老障碍。然而最近研究表明，肿瘤细胞在一定条件下可以被诱导而进入衰老状态，例如低于凋亡诱导剂量的DNA损伤因子即可引起肿瘤细胞发生加速性衰老，也称为早衰。也有研究表明，衰老是人体防止正常细胞向恶性细胞转化的重要机制之一。食管癌是我国高发恶性肿瘤之一，其预后差，病死率高，成为严重威胁人类生命健康的疾病之一。然而究竟哪些基因参与了食管正常细胞向恶性细胞转化的过程仍不是十分清楚，衰老机制在其中所发挥的作用至今也未见有研究报道。本实验室在前期研究中发现GRP78基因作为新的衰老相关调控基因，与P53等衰老调控基因有着密切联系。而异染色质相关蛋白1(heterochro-Matin-associated protein 1，HP1)参与组成了染色质相关多聚蛋白复合物，并且作为哺乳动物细胞的转录抑制因子，可引起稳定的转录沉默效应。其中HP1-γ作为衰老相关异染色质灶形成的标志蛋白，已被广泛用于标记组织和细胞衰老。传统的光镜下分析免疫组化结果的方法，由于是阅片者主观对染色强度或面积进行评分，其结果往往存在着客观性不足和代表性不强等缺陷。激光扫描细胞仪(Laser Scanning Cytometer，LSC)通过多组参数分析细胞及形态，主要应用于定量细胞内物质及组织扫描、多时段同点分析等。由于其可一次性对整张切片上所选区域进行扫描，并能快速输出客观、准确的分析结果，因此近年来逐渐被用于组织芯片染色结果的分析。基于以上研究背景和研究基础，我们以包含食管癌各级病变标本的组织芯片作为研究对象，通过激光扫描细胞仪扫描分析GRP78蛋白和HP1-γ蛋白的免疫组化结果，初步探讨GRP78蛋白和HP1-γ蛋白在食管癌恶性转化过程中的表达及相关关联情况。
方法
选用含各级别食管癌病变组织的组织芯片，采用免疫组化方法检测其中的GRP78蛋白和HP1-γ蛋白的表达，并通过激光扫描细胞仪对免疫组化结果进行定量分析。GRP78蛋白和HP1-γ蛋白的表达相关性采用卡方检验进行分析。
结果
激光扫描细胞仪扫描整张组织芯片后获得选定组织区域的虚拟图及阳性像素点百分比；正常食管组织、中．重度异型增生、原位癌、食管鳞癌的GRP78阳性率分别为0％(0／8)，90.4％(19／21)、83.3％(5／6)和47.4％(18／38)。GRP78在正常食管、中-重度异型增生加原位癌、食管鳞癌三组中的表达在统计学上有显著性差异(P＜0.01)。HP1-γ蛋白主要在胞核表达。HP1-γ蛋白在正常食管组织、中-重度异型增生、原位癌、食管鳞癌的阳性率分别为37.5％(3／8)，100％(21／21)、100％(6／6)和23.7％(9／38)。HP1-γ在正常食管、中-重度异型增生加原位癌、食管鳞癌这三组组织中表达在统计学上有显著性差异(P＜0.01)。GRP78蛋白和HP1-γ蛋白的表达具有高度相关性(P＜0.01)。
结论
激光扫描细胞仪用于免疫组化结果的分析具有高通量、高度客观、准确的优点。衰老可能是机体阻止正常食管细胞向恶性细胞转化的重要机制。GRP78蛋白在食管癌癌前病变中呈高表达且与HP1-γ具有高度相关性，提示其可能在机体通过衰老抵抗恶性转化的过程中扮演某种重要角色。
Background and Objective
So far many kinds of human cell lines and zooblasts can be cultured in vitro. Moreover,the reproductive activity of most cultured cells was limited. When cells proliferated into acertain stage they would not divide any more, which meant senescence. Gradually peoplediscovered that senescence existed in many human cell lines and cell lines of other specieswhen Hayflick discovered the phenomenon in human fibroblast in 1960's. The local cellswould proliferated abnormally and carcinogenesis when the body received all kinds ofharmful stimulation. Through continuous study on senescence people gradually had a newrealization about the relation between senescence and tumor, that the anti-tumor effect ofsenescence was an important anti-tumor mechanism in the body. Many studies in vitro hasverified that the carcinogenesis was inhibited by the senescence pathway when cells werestimulated by oncogene, more studies in vivo also verified it. Nonetheless, many cellswhich were transforming into malignant cells could bypass the inhibition of senescence andchange into tumor cells ultimatedly owing to the senescence barriers. In vitro and in vivostudies has established that tumour cells can be induced into accelerated senescence bymany drugs which could cause DNA damage, such as cisplatin, camptothecin and VP16.And some studies have established that some senescence regulating genes in the ATMrelated DNA damage pathway take part in the accelerated senescence induced bychemotherapy. But the concrete mechanism is still unclear. Therefore in our study wechoose cisplatin which is the most common chemotherapy drug in clinic to induceNG108-15, Hela, A2780 cell lines respectively, which are different organization types. Theobjection is to make it clear that what roles these senescence regulating genes play in thesenescence process.
Methods
1. Apply cisplatin with different concentration gradient to affect tumour cells, choose theconcentration when the senescence rate was highest and without obvious apoptosis.
2. Apply cell immunofluorescence to test the expression of HP1-γprotein during theprocess of cell senescence. Apply SA-βGal staining method to test the senescence oftumour cells induced by cisplatin.
3. Apply MTT and CFSE staining method to test the proliferation of tumour cells duringsenescence.
4. Apply caffeine to inhibit ATM, Apply PI staining method to test the cell cycle byFACS. Apply western blot to test the expression of senescence regulating genes such asP53, P21 and Cdc2.
Results
All the NG108-15、Hela、A2780 cell lines showed senescence phenotype when theywere induced by certain low dose cisplatin. The senescent cells become large and flatten,increased vacuolus in cytoplasm, positive staining of SA-β-Gal, HP1-γexpression increasedand gathered in the nuclear as foci. The senescent cells stopped proliferating and weremainly blocked during G2/M period. Caffeine, as one of the ATM inhibitor, could make thetumour cells resistant to the senescence induced by cisplatin. The expression and activity ofP53, P21 and CDC2 genes changed obviously during the senescence process.
Conclusion
NG108-15, Hela and A2780 cells could be induced into accelerated senescence by acertain low dose cisplatin. The DNA damage pathway genes such as ATM, P53, P21 andCDC2 have taken part in the senescence process. The senescence induced by cisplatincould be reversed by inhibition of ATM.
Background and Objective
Two-dimensional gel electrophoresis proteomics technology is the most widely usedprotein separation technique in the study of proteomics up to now. Through isoelectriccondensation in the first dimension, different proteins concentrate in accordance with theirisoelectric point, and then the proteins of the same isoelectric point separate in accordancewith their molecular weight in the second dimension of the SDS-PAGE gel. On the basis ofabove theorem, two-dimensional gel electrophoresis proteomics technology can separatelarge amounts of proteins according their isoelectric point and molecular weight one time.Respecting its advantage of high-flux, large amounts of researches have studied relevantprotein in the tumorigenesis and progression of carcinoma using this technique. After theeffect of different kinds of factors, cells change from the state of active proliferative into thestate of irreversible growth arrest, which is named cell senescence. Tumor cells are generallyconsidered that they may have obstacles in the senescence pathway. However, recentresearch has shown that some DNA damage factors(such as cisplatin) can induce tumorcell senescence both in vivo and in vitro experiments. Nevertheless, it is still unknown thatwhich genes participate in the process of cell senescence. So we systematically screenrelevant differential expression protein in the cisplatin induced tumor senescent cell throughtwo-dimensional gel electrophoresis proteomics technology, in order to find some newsenescence-associate genes.
Methods
1. Using certain dose of cisplatin induced NG108-15 cell senescence, and then extractedtotal protein.
2. Screened differential expression protein between NG108-15 normal cell and NG108-15 senescent cell through two-dimensional gel electrophoresis proteomics technology.Repeated the experiment three times and selected the spots whose difference was more than1.5 times for the follow-up analysis.
3. Identified the selected protein by MALDI-TOF-MS.
4. Verified expression of the selected protein screened by two-dimensional gelelectrophoresis proteomics technology through Western blot and Cell immunofluorescence;Constructed subcutaneous tumor model in nude mouse and then injected cisplatin in tumorsite. Detected the expression of GRP78 through immunohistochemitry and observed thesituation of tumor cell senescence through SA-β-Gal stainning.
Results
A total of 517±25 sports were detected in control group, while there were 474±21sports detected in NG108-15 senescent cell through two-dimensional gelelectrophoresis. Selected 5 significantly differential expression protein sports for thefollow-up analysis. After MALDI-TOF MS analysis, a total of 5 differential expressionprotein were detected. The results of western blot detecting the expression of Vimentin andGRP78 were coincidence with that of two-dimensional gel electrophoresis. GRP78 weredetected that it had a lower expression in cisplatin induced NG108-15 senescent cell bywestern blot, cell immunofluorescence and subcutaneous tumor model in nude mouse.
Conclusion
As one of effective technique for selecting the differential expression protein,wo-dimensional gel electrophoresis can be successfully used for screening newcisplatin-induced senescence-associated gene. The expression of GRP78 decreasedsignificantly in the process of cisplatin-induced NG108-15 cell senescence and it may havesome link with cisplatin-induced cell senescence.
Backgroud and objection
Glucose regulated protein 78(GRP78), which is also named the immu-noglobulinheavy chain binding protein(Bip), has high homology with GRP78 family. It is generallybelieved that GRP78 is one member of the GRP78 family. GRP78 takes part in manyphysiology and pathology process of cells, which has complicated biological function. It isnot only a molecular chaperon participating protein folding and transferring, but alsoup-regulated to maintain the stabilization of ER and protect cells as an ER stress proteinwhen low carbohydrates, hypoxia and low calcium. In recent years, many studiesestablished that GRP78 synthesis was high in many tumour cells which can endue them theanti-apoptosis characterization, and affect antigenicity of tumour cells. On the other hand,recent study show that GRP78 highly expressed in the ER of astrocyte exposed to heavymetal environment, which can protect sensitive neuron. Tumor is a age associated diseasewhich often occurred in old people. In 1961 Hayflick discovered that there was limitedproliferation in the culture cells in vitro, which was called cell senescence. So far thestudies about cell senescence often referred to cell cycle, free radical, relation betweenDNA damage and senescence, telomere theory, mitochondria and senescence, senescenceassociated genes and long life genes, cell signal transduction and so on. Nonetheless, therelation between GRP78 and senescence has not been seen in literature. Our previous workhas established by 2DE that GRP78 decreased obviously during senescence of NG108-15cells, which may be a new senescence regulating gene associated with cisplatin induction.Therefore, we are going to approach the role of GRP78 played during senescence inducedby cisplatin and to elucidate the regulating mechanism, which may afford clinic therapynew target and effective plan.
Methods
1. Western blot test the GRP78 expression during senescence induced by cisplatin inNG108-15, Hela, A2780 cells.
2. GRP78 was transfected into tumour cells by lipofection. Apply Western blot to test thetransfection effect and expression of associated senescence genes. The senescencesensitivity for tumour cells induced by cisplatin after GRP78 transfection was detectedby SA-β-Gal staining method.
3. Apply GRP78 inductor 2DG or A23187 to up-regulate GRP78 in different tumour cells.The sensitivity of senescence of tumour cells induced by cisplatin after GRP78up-regulation was detected by SA-β-Gal staining method.
4. Apply laser confocal microscopy to test the relative concentration of calcium inkytoplasm, apply topoismerase inhibitor camptothecin and adriamycin to induce tumourcells senescence, senescence of tumour cells induced by different drugs were detectedby SA-β-Gal staining method. Apply Western blot to test GRP78 expression during thesenescence induced by different drugs.
5. Apply immune histochemistry to test GRP78 and HP1-γexpression in cervical cancerand ovary cancer.
Results
1. GRP78 expression decreased during senescence induced by cisplatin in NG108-15,Hela, A2780 cells. CHOP and Caspase 7 expression also had a little decrease, whichmeant that there was no ER stress associated apoptosis occurred during senescence.
2. P53 protein expression increased obviously when GRP78 was knock down by SiRNAin NG108-15, Hela, A2780 cells.
3. The senescence sensitivity of NG108-15 and Hela cells induced by cisplatin had a littleincrease. But there was no positive staining of SA-β-Gal in A2780 cell after GRP78knockdown.
4. The senescence sensitivity of NG108-15 decreased obviously when GRP78 wasup-regulated by 2DG, Which can be reversed by GRP78 knockdown after 2DGinduction. Nonetheless, the phenomenon did not exist in Hela and A2780 cells. Thesenescence sensitivity of Hela and A2780 cells decreased obviously when GRP78 wasup-regulated by A23187, Which can be reversed by GRP78 knockdown after A23187induction.
5. It was obviously that increase of P53, P21 and P-CDC2 expression tendency duringcisplatin induction was slow down by GRP78 up-regulation.
6. The calcium concentration of kytoplasm increased after24h' cisplatin induction. But ifGRP78 was up-regulated by inductor, there was no change of hytoplasm calciumconcentration after24h' cisplatin induction. If GRP78 was knocked down afterup-regulation, the calcium concentration of kytoplasm can increase again with cisplatin.
7. For immunohistochemistry results, GRP78 expressed highly in normal cervical tissuesand precancerous change and cancer in situ, but express lowly in cancer tissues. GRP78expression was higher in ovarian cancer than in normal and benign tissues. GRP78expression had high dependability with HP1-γexpression in these two cancers.
Conclusion
1. As a new discovered chemotherapy induced-senescence associated regulating gene,GRP78 has rivalry and promotion with DNA damage-repair genes such as P53, P21 andCdc2. Down-regulation of GRP78 could promote the senescence induced by cisplatin.Nonetheless, up-regulation of GRP78 can decrease sensitivity of cisplatin-inducedsenescence. The phenomenon demonstrated that GRP78 may play a resistant role insenescence induced by cisplatin.
2. GRP78 may affect senescence sensitivity of tumour cells induced by cisplatin throughinfluencing DNA damage-repair pathway associated genes and change of kyroplasmcalcium concentration. GRP78 expression was higher in cervical precancerosis thanthat in normal tissues and cancer tissues. Moreover, GRP78 expression was highly relevant to HP1-γexpression, which meant that GRP78 may participate in thetransforming process from normal cells to malignant cells. GRP78 highly expressed inovarian cancer, which meant that it may influence chemotherapy effect of ovariancancer through anti-senescence.
Background and objective
In 1961, Hayflick discovered that there was limited proliferating capability in culturedcell in vitro. He named it as cell senescence. Tumor cells showed to be immortalized andcan proliferate unlimitedly. So tumor cell were considered to have a certain senescenceblock.. Nonetheless, recent studies established that tumor cells can be induced to besenescent under some conditions. For example, lower than apoptosis-induced dose DNAdamage agent can induce cell to have accelerated senescence, which was also calledpremature senescence. Other studies showed that senescence was an important mechanismprotecting normal cell from malignant transforming.
Esophageal carcinoma is one of the most common malignant tumor severily harmingpeople's life in our country. Esophageal carcinoma has a worse progonosis, nonetheless,which genes participate in its carcinogenesis and progression is still unknown. What rolethe senescence mechanism played in this process is also unclear. GRP78 gene was found tobe closely associated with senescence associated genes such as P53, which was discoveredas a new senescence regulating gene in our previous work. As one of the transcriptioninhibition factors, Heterochro-Matin-associated protein 1(HP1) is a component of thepolyprotein complex which could give rise to gene silencing. As a mark of senescenceassociated heterochromatin foci, HP1-γhas been widely used to show the cell and tissuesenescence. Owing to accessing paraffin section subjectively by staining intensity or area,the analysis results of traditional scoring method under light microscopy is lack of objectivity, which representativeness is not strong. Laser Scanning Cytometer(LSC)analyzes cells and morphous by sets of parameters, therefore it has been not only mainlyapplied to scan intracelluar materials and tissue quantitively, but also analyze the samepoint at different period of time. Because it can scan the selected region of entire paraffinsection at one time and can export objective and accurate result rapidly, LSC has beengradually used to analyze the immunohistochemistry results of tissue microarray. Based onstudy background and previous work above, we selected a tissue microarray containingdifferent grades of esophageal carcinoma and detected the expression of GRP78 and HP1-γprotein by immunohistochemistry. The results of immunohistochemistry were analyzedquantitatively by using laser scanning cytometer. We initially approach GRP78 andHP1-γexpression during malignant transformation of esophageal cell and their correlation.
Methods
A tissue microarray containing different grades of esophageal carcinoma wasselected. GRP78 and HP1-γprotein was detected by immunohistochemistry. The results ofimmunohistochemistry were analyzed quantitatively by laser scanning cytometer. Thecorrelation of GRP78 expression and HP1-γexpression was analyzed by chi square test.
Results
The phantoms and positive pixel percentage of selected areas were obtained afterscanning the entire tissue microarray. The positive rates of normal esophagus,moderate-severe atypical hyperplasia, carcinoma in site and squamous carcinoma were0%(0/8), 90.4%(19/21), 83.3%(5/6) and 47.4%(18/38), respectively. There was statisticaldiscrepancy of GRP78 expression among normal esophagus, moderate-severe atypicalhyperplasia plus carcinoma in site and squamous carcinoma(P＜0.01). HP1-γprotein wasprimarily expressed in the cell nucleus. The positive rate of normal esophagus,moderate-severe atypical hyperplasia, carcinoma in site and squamous cancer were 37.5%(3/8), 100% (21/21), 100%(6/6) and 23.7%(9/38), respectively. There was statistical discrepancy of HP1-γexpression among normal esophagus, moderate-severe atypicalhyperplasia plus carcinoma in site and squamous cancer(P＜0.01). There was highcorrelation between GRP78 expression and HP1-γexpression in esophageal carcinoma(P＜0.01).
Conclusion
There are many advantages such as high resolution, high objectivity and accuracywhen using laser scanning cytometer to analyze the immunohistochemistry results.Senescence may be an important mechanism for prevent normal cells from malignanttransformation. GRP78 highly expressed in precancerosis of esophageal and its expressionwas extremely correlated with HP-γexpression. These results demonstrate that GRP78 mayplay an important role in protecting normal cells from malignant transformation withsenescence.
迄今为止，许多类型的人类和动物细胞都能够体外培养，然而大部分培养细胞的增殖能力都有一定限度，当细胞增殖到一定阶段后就会进入不分裂的阶段，也就意味着进入了衰老(senescence)。自Hayflick上世纪60年代从人类纤维原细胞中发现了这种现象后，人们逐渐发现人类其它各类细胞和其它物种的细胞也存在着衰老。当人体受到各种有害因素刺激时，局部组织的细胞会产生异常增生从而形成肿瘤。通过对细胞衰老的不断研究，人们逐渐对衰老与肿瘤关系有了一种新的认识，认为细胞衰老的抗肿瘤作用是机体对抗肿瘤的一种重要机制。有很多体外试验表明，细胞受癌基因刺激后是通过发生衰老这一途径来抑制肿瘤形成的，更多的体内研究也证实了这一作用。然而，由于细胞衰老机制障碍，还是有很多正在发生恶性转化的细胞绕过衰老抑制而最终成为恶性肿瘤细胞。现有体内和体外的研究证实一些具有DNA损伤作用的化疗药物如顺铂、喜树碱、依托铂苷等，能够引起肿瘤细胞的加速性衰老(acceleratedsenescence)。也有一些研究证实ATM基因相关的DNA损伤修复通路中的衰老相关调控基因参与了化疗诱导的加速性衰老的发生，但是其具体机制仍不是十分清楚。因此本研究采用顺铂这一临床上最常用的化疗药物分别诱导NG108-15，Hela，A2780三株不同组织类型的肿瘤细胞系，试图阐明相关衰老调控基因在其中所发挥的作用。
方法
一、应用不同浓度梯度顺铂作用肿瘤细胞，选择能够达到最高衰老率而无明显凋亡发生的浓度作为实验浓度。
二、运用细胞免疫荧光检测肿瘤细胞衰老过程中HP1-γ蛋白(衰老相关异染色质灶)的表达变化；应用SA-βGal(衰老相关β-半乳糖苷酶)染色法检测顺铂诱导肿瘤细胞衰老情况。
三、采用MTT法和CFSE染色法检测顺铂诱导肿瘤细胞衰老过程中的细胞增殖情况；
四、选用咖啡因抑制ATM基因；PI单染流式细胞仪检测细胞周期变化；Western blot检测P53、P21、CDC2等衰老相关调控基因表达变化情况；
结果
在一定小剂量顺铂诱导下，NG108-15、Hela、A2780细胞均呈现出衰老表型。细胞变大变扁平，胞浆空泡增多，SA-βGal染色呈阳性着色，HP1-γ蛋白表达增多并呈灶状聚集分布在细胞核。衰老细胞增殖几乎停滞，细胞周期主要阻滞在G2／M期。ATM抑制剂咖啡因可以抵抗顺铂诱导的衰老发生。P53、P21、CDC2基因在顺铂诱导衰老过程中表达量和活性明显发生改变。
结论
一定小剂量的顺铂可以诱导NG108-15、Hela、A2780细胞发生加速性衰老。DNA损伤修复通路上的ATM、P53、P21、CDC2等基因参与了顺铂诱导的肿瘤细胞衰老过程；抑制ATM基因表达后可以逆转顺铂诱导的肿瘤细胞衰老。
第二部分利用二维电泳技术筛选新的顺铂诱导衰老相关基因
研究背景与目的
二维电泳技术是迄今为止蛋白质组学研究中使用最广泛的一种蛋白质分离技术，通过在第一向的等电聚焦，不同的蛋白质依照其等电点的大小聚集，继而在第二向的SDS-PAGE凝胶电泳将相同等电点的蛋白质依照其分子量大小的不同而进行分离。根据以上原理，二维电泳能够一次性将大量的不同等电点、不同分子量的蛋白质进行分离。鉴于二维电泳技术的高通量的优势，已有相当多的研究采用该技术研究肿瘤发生、发展过程中的相关蛋白质。细胞衰老指细胞从具有活性增殖的状态通过各种因素的影响而成为不可逆转的生长停滞状态。肿瘤细胞通常被认为存在衰老通路障碍。然而最近的研究证实无论是在体内或是体外实验中，一些DNA损伤因子如顺铂等化疗药物可以诱导肿瘤细胞发生衰老。然而究竟有哪些基因参与其中，这些基因与衰老的关系如何等均不是十分清楚。因此本实验选取二维电泳技术作为主要研究手段，系统筛选分析顺铂诱导肿瘤细胞衰老过程中相关差异表达蛋白，以期找到新的衰老相关调控基因。
方法
一、以一定剂量顺铂诱导NG108-15细胞衰老，提取细胞总蛋白；
二、采用二维电泳法筛选NG108-15细胞衰老前后表达差异蛋白。实验重复3次，选取表达差异在1.5倍以上的蛋白点进行后续分析。
三、MALDI-TOF-MS质谱分析鉴定选取蛋白质
四、Western blot、细胞免疫荧光验证二维电泳筛选出的差异蛋白的表达情况；构建裸鼠皮下瘤模型，瘤根原位注射顺铂。冰冻切片免疫组化法检测GRP78蛋白表达变化情况，SA-β-Gal染色法检测瘤体细胞衰老情况；
结果
对照SDS-PAGE胶中共有517±25个点被检测出，衰老NG108-15细胞对应的胶中共有474±21点被检测出。选取二维电泳胶中NG108-15细胞衰老前后5个表达差异明显的蛋白点进行后续分析。MALDI-TOF MS质谱分析共检测出5个差异蛋白。Western blot验证了Vimentin和GRP78在衰老前后的表达变化与二维电泳结果一致。Western blot、细胞免疫荧光和荷瘤裸鼠模型均证实GRP78在顺铂诱导NG108-15细胞发生衰老后表达明显降低。
结论
作为高通量筛选差异蛋白的有效技术手段，二维电泳技术可被成功用于筛选新的顺铂诱导肿瘤细胞衰老相关调控基因。GRP78蛋白在顺铂诱导NG108-15细胞衰老的过程中表达明显降低，可能与顺铂诱导的衰老存在某种联系。
第三部分GRP78基因在顺铂诱导肿瘤细胞衰老过程中的功能鉴定
研究背景与目的
葡萄糖调节蛋白78(glucose regulated p rotein，GRP78)，通常也被称为免疫球蛋白重链结合蛋白(the immu-noglobulin heavy chain binding p rotein，Bip)，与热休克蛋白70家族有非常高的同源性，一般认为它是热休克蛋白70家族中的一种蛋白质。GRP78参与了细胞的许多生理和病理过程，其生物学功能很复杂。它既是一种分子伴侣在蛋白质的折叠和转运中起着重要的作用，又是定位于内质网上的一种应激蛋白，在低糖、低氧、低Ca2 +等应激状态下其表达明显升高，进而保护细胞免受损伤。近年来研究表明GRP78在某些肿瘤细胞中表达较高，能够赋予肿瘤细胞抗凋亡的特性，并能够影响肿瘤细胞的抗原性。最近研究认为当星型胶质细胞受到重金属刺激的时候会高表达GRP78，从而达到保护神经元细胞免受损害的效果。肿瘤多发生于年龄较大的人群，与年龄存在着一定的相关性。1961年Hayflick在对体外培养细胞研究中发现细胞的增值并不是无限的，也有一定的限制性，他把这种现象叫做细胞衰老。迄今为止对细胞衰老机制方面的研究通常包括对细胞周期、自由基及DNA损伤与衰老的关系、衰老的端粒学说、线粒体与衰老、衰老相关基因和长寿基因、细胞信号转导等方面的研究。然而有关GRP78与衰老之间关系的研究还未见文献报道。本实验室前期通过二维电泳等技术证明GRP78基因在顺铂诱导NG108-15细胞衰老过程中表达明显降低，其可能是新的顺铂诱导肿瘤细胞衰老的相关调控基因。因此本研究拟对GRP78基因在顺铂诱导肿瘤细胞衰老中所扮演的角色进行深入探讨，以期阐明相关调控机制，为临床化疗提供新的治疗靶点和有效方案。
方法
一、Western blot分别检测GRP78在顺铂诱导NG108-15、Hela、A2780细胞衰老过程中的表达变化情况；
二、SiGRP78通过脂质体法转染肿瘤细胞，Western blot验证转染效果并检测相关衰老调控基因变化情况。SA-β-Gal染色检测封闭GRP78后肿瘤细胞对顺铂诱导衰老的敏感性变化情况；
三、应用GRP78诱导剂2DG和A23187上调GRP78在不同肿瘤细胞系的表达，SA-β-Gal染色检测上调GRP78蛋白后肿瘤细胞对顺铂诱导衰老的敏感性变化情况；Western blot检测上调GRP78后顺铂诱导衰老过程中P53、P21等衰老相关调控基因表达变化情况；
四、应用激光共聚焦显微镜检测细胞胞浆内钙离子浓度相对变化情况；
五、免疫组化法检测GRP78蛋白及HP1-γ蛋白在宫颈癌和卵巢癌中的表达情况。
结果
一、Western blot结果显示GRP78在NG108-15、Hela、A2780细胞顺铂诱导衰老过程中表达降低。Western blot结果显示CASPASE7在衰老过程中始终以无活性形式存在，提示在顺铂诱导肿瘤细胞衰老过程中无内质网凋亡途径激活。
二、在NG108-15、Hela、A2780细胞中利用GRP78SiRNA封闭GRP78表达后，P53表达均明显升高；
三、在NG108-15、Hela和A2780细胞中利用SiRNA下调GRP78表达后细胞对顺铂诱导的衰老敏感性略有增加。利用SiRNA下调卵巢癌顺铂耐药细胞C13K的GRP78表达后衰老相关β半乳糖苷酶染色阳性率明显升高。
四、2DG诱导GRP78表达上调后NG108-15细胞对顺铂诱导衰老的敏感性明显降低，接着SiRNA下调GRP78表达后敏感性恢复。而在Hela和A2780细胞中则无明显改变。在Hela和A2780细胞中A23187诱导GRP78表达上调后细胞对顺铂诱导衰老的敏感性显著降低，SiRNA下调GRP78表达后敏感性恢复。
五、Western blot结果显示上调GRP78表达后顺铂诱导肿瘤细胞衰老过程中P53表达升高趋势明显被抑制。
六、胞浆内钙离子荧光强度检测显示诱导衰老剂量的顺铂作用NG108-15细胞24小时后胞浆内钙离子浓度升高，而上调GRP78后顺铂作用24小时胞浆内钙离子浓度无明显升高。诱导剂诱导GRP78高表达后接着用SiRNA下调GRP78表达，则顺铂作用24小时时胞浆内钙离子浓度显著升高。
七、免疫组化结果显示GRP78在宫颈癌的正常组织以及癌前病变和原位癌中呈高表达，而在癌组织中的表达较低。GRP78在卵巢正常和良性组织中表达较低，而在卵巢癌中呈高表达。在各种肿瘤组织GRP78表达与HP1-γ表达具有高度相关性。(P＜0.05)
结论
一、GRP78作为新发现的化疗介导的衰老相关调控基因，与P53、P21、CDC2等DNA损伤修复基因具有一定的相互拮抗或促进作用；下调GRP78的表达能够显著提高P53基因的表达水平，而上调其表达则能够显著降低肿瘤细胞对顺铂诱导衰老的敏感性。提示GRP78在顺铂诱导肿瘤衰老过程中主要扮演抵抗衰老的角色。
二、GRP78可能通过影响DNA损伤修复通路基因的表达及胞浆内钙离子浓度的改变从而影响肿瘤细胞对顺铂诱导衰老的敏感性。在宫颈癌癌前病变中GRP78的表达明显高于正常组织和癌组织，且与HP1-γ蛋白的表达具有很强的关联性，提示其可能参与了正常细胞向癌细胞恶性转化的过程。在卵巢癌中GRP78呈高表达，提示其可能通过抗衰老途径影响卵巢癌化疗效果。
第四部分应用激光扫描细胞仪分析GRP78蛋白和HP1-γ蛋白在食管癌各级病变中的表达情况及两者表达相关性的研究
研究背景和目的
1961年Hayflick在对体外培养细胞研究中发现细胞有增殖极限的现象，并称之为细胞衰老。以往的观点认为，肿瘤细胞具有无限制增殖的永生化能力，因此被认为存在一定的衰老障碍。然而最近研究表明，肿瘤细胞在一定条件下可以被诱导而进入衰老状态，例如低于凋亡诱导剂量的DNA损伤因子即可引起肿瘤细胞发生加速性衰老，也称为早衰。也有研究表明，衰老是人体防止正常细胞向恶性细胞转化的重要机制之一。食管癌是我国高发恶性肿瘤之一，其预后差，病死率高，成为严重威胁人类生命健康的疾病之一。然而究竟哪些基因参与了食管正常细胞向恶性细胞转化的过程仍不是十分清楚，衰老机制在其中所发挥的作用至今也未见有研究报道。本实验室在前期研究中发现GRP78基因作为新的衰老相关调控基因，与P53等衰老调控基因有着密切联系。而异染色质相关蛋白1(heterochro-Matin-associated protein 1，HP1)参与组成了染色质相关多聚蛋白复合物，并且作为哺乳动物细胞的转录抑制因子，可引起稳定的转录沉默效应。其中HP1-γ作为衰老相关异染色质灶形成的标志蛋白，已被广泛用于标记组织和细胞衰老。传统的光镜下分析免疫组化结果的方法，由于是阅片者主观对染色强度或面积进行评分，其结果往往存在着客观性不足和代表性不强等缺陷。激光扫描细胞仪(Laser Scanning Cytometer，LSC)通过多组参数分析细胞及形态，主要应用于定量细胞内物质及组织扫描、多时段同点分析等。由于其可一次性对整张切片上所选区域进行扫描，并能快速输出客观、准确的分析结果，因此近年来逐渐被用于组织芯片染色结果的分析。基于以上研究背景和研究基础，我们以包含食管癌各级病变标本的组织芯片作为研究对象，通过激光扫描细胞仪扫描分析GRP78蛋白和HP1-γ蛋白的免疫组化结果，初步探讨GRP78蛋白和HP1-γ蛋白在食管癌恶性转化过程中的表达及相关关联情况。
方法
选用含各级别食管癌病变组织的组织芯片，采用免疫组化方法检测其中的GRP78蛋白和HP1-γ蛋白的表达，并通过激光扫描细胞仪对免疫组化结果进行定量分析。GRP78蛋白和HP1-γ蛋白的表达相关性采用卡方检验进行分析。
结果
激光扫描细胞仪扫描整张组织芯片后获得选定组织区域的虚拟图及阳性像素点百分比；正常食管组织、中．重度异型增生、原位癌、食管鳞癌的GRP78阳性率分别为0％(0／8)，90.4％(19／21)、83.3％(5／6)和47.4％(18／38)。GRP78在正常食管、中-重度异型增生加原位癌、食管鳞癌三组中的表达在统计学上有显著性差异(P＜0.01)。HP1-γ蛋白主要在胞核表达。HP1-γ蛋白在正常食管组织、中-重度异型增生、原位癌、食管鳞癌的阳性率分别为37.5％(3／8)，100％(21／21)、100％(6／6)和23.7％(9／38)。HP1-γ在正常食管、中-重度异型增生加原位癌、食管鳞癌这三组组织中表达在统计学上有显著性差异(P＜0.01)。GRP78蛋白和HP1-γ蛋白的表达具有高度相关性(P＜0.01)。
结论
激光扫描细胞仪用于免疫组化结果的分析具有高通量、高度客观、准确的优点。衰老可能是机体阻止正常食管细胞向恶性细胞转化的重要机制。GRP78蛋白在食管癌癌前病变中呈高表达且与HP1-γ具有高度相关性，提示其可能在机体通过衰老抵抗恶性转化的过程中扮演某种重要角色。
Background and Objective
So far many kinds of human cell lines and zooblasts can be cultured in vitro. Moreover,the reproductive activity of most cultured cells was limited. When cells proliferated into acertain stage they would not divide any more, which meant senescence. Gradually peoplediscovered that senescence existed in many human cell lines and cell lines of other specieswhen Hayflick discovered the phenomenon in human fibroblast in 1960's. The local cellswould proliferated abnormally and carcinogenesis when the body received all kinds ofharmful stimulation. Through continuous study on senescence people gradually had a newrealization about the relation between senescence and tumor, that the anti-tumor effect ofsenescence was an important anti-tumor mechanism in the body. Many studies in vitro hasverified that the carcinogenesis was inhibited by the senescence pathway when cells werestimulated by oncogene, more studies in vivo also verified it. Nonetheless, many cellswhich were transforming into malignant cells could bypass the inhibition of senescence andchange into tumor cells ultimatedly owing to the senescence barriers. In vitro and in vivostudies has established that tumour cells can be induced into accelerated senescence bymany drugs which could cause DNA damage, such as cisplatin, camptothecin and VP16.And some studies have established that some senescence regulating genes in the ATMrelated DNA damage pathway take part in the accelerated senescence induced bychemotherapy. But the concrete mechanism is still unclear. Therefore in our study wechoose cisplatin which is the most common chemotherapy drug in clinic to induceNG108-15, Hela, A2780 cell lines respectively, which are different organization types. Theobjection is to make it clear that what roles these senescence regulating genes play in thesenescence process.
Methods
1. Apply cisplatin with different concentration gradient to affect tumour cells, choose theconcentration when the senescence rate was highest and without obvious apoptosis.
2. Apply cell immunofluorescence to test the expression of HP1-γprotein during theprocess of cell senescence. Apply SA-βGal staining method to test the senescence oftumour cells induced by cisplatin.
3. Apply MTT and CFSE staining method to test the proliferation of tumour cells duringsenescence.
4. Apply caffeine to inhibit ATM, Apply PI staining method to test the cell cycle byFACS. Apply western blot to test the expression of senescence regulating genes such asP53, P21 and Cdc2.
Results
All the NG108-15、Hela、A2780 cell lines showed senescence phenotype when theywere induced by certain low dose cisplatin. The senescent cells become large and flatten,increased vacuolus in cytoplasm, positive staining of SA-β-Gal, HP1-γexpression increasedand gathered in the nuclear as foci. The senescent cells stopped proliferating and weremainly blocked during G2/M period. Caffeine, as one of the ATM inhibitor, could make thetumour cells resistant to the senescence induced by cisplatin. The expression and activity ofP53, P21 and CDC2 genes changed obviously during the senescence process.
Conclusion
NG108-15, Hela and A2780 cells could be induced into accelerated senescence by acertain low dose cisplatin. The DNA damage pathway genes such as ATM, P53, P21 andCDC2 have taken part in the senescence process. The senescence induced by cisplatincould be reversed by inhibition of ATM.
Background and Objective
Two-dimensional gel electrophoresis proteomics technology is the most widely usedprotein separation technique in the study of proteomics up to now. Through isoelectriccondensation in the first dimension, different proteins concentrate in accordance with theirisoelectric point, and then the proteins of the same isoelectric point separate in accordancewith their molecular weight in the second dimension of the SDS-PAGE gel. On the basis ofabove theorem, two-dimensional gel electrophoresis proteomics technology can separatelarge amounts of proteins according their isoelectric point and molecular weight one time.Respecting its advantage of high-flux, large amounts of researches have studied relevantprotein in the tumorigenesis and progression of carcinoma using this technique. After theeffect of different kinds of factors, cells change from the state of active proliferative into thestate of irreversible growth arrest, which is named cell senescence. Tumor cells are generallyconsidered that they may have obstacles in the senescence pathway. However, recentresearch has shown that some DNA damage factors(such as cisplatin) can induce tumorcell senescence both in vivo and in vitro experiments. Nevertheless, it is still unknown thatwhich genes participate in the process of cell senescence. So we systematically screenrelevant differential expression protein in the cisplatin induced tumor senescent cell throughtwo-dimensional gel electrophoresis proteomics technology, in order to find some newsenescence-associate genes.
Methods
1. Using certain dose of cisplatin induced NG108-15 cell senescence, and then extractedtotal protein.
2. Screened differential expression protein between NG108-15 normal cell and NG108-15 senescent cell through two-dimensional gel electrophoresis proteomics technology.Repeated the experiment three times and selected the spots whose difference was more than1.5 times for the follow-up analysis.
3. Identified the selected protein by MALDI-TOF-MS.
4. Verified expression of the selected protein screened by two-dimensional gelelectrophoresis proteomics technology through Western blot and Cell immunofluorescence;Constructed subcutaneous tumor model in nude mouse and then injected cisplatin in tumorsite. Detected the expression of GRP78 through immunohistochemitry and observed thesituation of tumor cell senescence through SA-β-Gal stainning.
Results
A total of 517±25 sports were detected in control group, while there were 474±21sports detected in NG108-15 senescent cell through two-dimensional gelelectrophoresis. Selected 5 significantly differential expression protein sports for thefollow-up analysis. After MALDI-TOF MS analysis, a total of 5 differential expressionprotein were detected. The results of western blot detecting the expression of Vimentin andGRP78 were coincidence with that of two-dimensional gel electrophoresis. GRP78 weredetected that it had a lower expression in cisplatin induced NG108-15 senescent cell bywestern blot, cell immunofluorescence and subcutaneous tumor model in nude mouse.
Conclusion
As one of effective technique for selecting the differential expression protein,wo-dimensional gel electrophoresis can be successfully used for screening newcisplatin-induced senescence-associated gene. The expression of GRP78 decreasedsignificantly in the process of cisplatin-induced NG108-15 cell senescence and it may havesome link with cisplatin-induced cell senescence.
Backgroud and objection
Glucose regulated protein 78(GRP78), which is also named the immu-noglobulinheavy chain binding protein(Bip), has high homology with GRP78 family. It is generallybelieved that GRP78 is one member of the GRP78 family. GRP78 takes part in manyphysiology and pathology process of cells, which has complicated biological function. It isnot only a molecular chaperon participating protein folding and transferring, but alsoup-regulated to maintain the stabilization of ER and protect cells as an ER stress proteinwhen low carbohydrates, hypoxia and low calcium. In recent years, many studiesestablished that GRP78 synthesis was high in many tumour cells which can endue them theanti-apoptosis characterization, and affect antigenicity of tumour cells. On the other hand,recent study show that GRP78 highly expressed in the ER of astrocyte exposed to heavymetal environment, which can protect sensitive neuron. Tumor is a age associated diseasewhich often occurred in old people. In 1961 Hayflick discovered that there was limitedproliferation in the culture cells in vitro, which was called cell senescence. So far thestudies about cell senescence often referred to cell cycle, free radical, relation betweenDNA damage and senescence, telomere theory, mitochondria and senescence, senescenceassociated genes and long life genes, cell signal transduction and so on. Nonetheless, therelation between GRP78 and senescence has not been seen in literature. Our previous workhas established by 2DE that GRP78 decreased obviously during senescence of NG108-15cells, which may be a new senescence regulating gene associated with cisplatin induction.Therefore, we are going to approach the role of GRP78 played during senescence inducedby cisplatin and to elucidate the regulating mechanism, which may afford clinic therapynew target and effective plan.
Methods
1. Western blot test the GRP78 expression during senescence induced by cisplatin inNG108-15, Hela, A2780 cells.
2. GRP78 was transfected into tumour cells by lipofection. Apply Western blot to test thetransfection effect and expression of associated senescence genes. The senescencesensitivity for tumour cells induced by cisplatin after GRP78 transfection was detectedby SA-β-Gal staining method.
3. Apply GRP78 inductor 2DG or A23187 to up-regulate GRP78 in different tumour cells.The sensitivity of senescence of tumour cells induced by cisplatin after GRP78up-regulation was detected by SA-β-Gal staining method.
4. Apply laser confocal microscopy to test the relative concentration of calcium inkytoplasm, apply topoismerase inhibitor camptothecin and adriamycin to induce tumourcells senescence, senescence of tumour cells induced by different drugs were detectedby SA-β-Gal staining method. Apply Western blot to test GRP78 expression during thesenescence induced by different drugs.
5. Apply immune histochemistry to test GRP78 and HP1-γexpression in cervical cancerand ovary cancer.
Results
1. GRP78 expression decreased during senescence induced by cisplatin in NG108-15,Hela, A2780 cells. CHOP and Caspase 7 expression also had a little decrease, whichmeant that there was no ER stress associated apoptosis occurred during senescence.
2. P53 protein expression increased obviously when GRP78 was knock down by SiRNAin NG108-15, Hela, A2780 cells.
3. The senescence sensitivity of NG108-15 and Hela cells induced by cisplatin had a littleincrease. But there was no positive staining of SA-β-Gal in A2780 cell after GRP78knockdown.
4. The senescence sensitivity of NG108-15 decreased obviously when GRP78 wasup-regulated by 2DG, Which can be reversed by GRP78 knockdown after 2DGinduction. Nonetheless, the phenomenon did not exist in Hela and A2780 cells. Thesenescence sensitivity of Hela and A2780 cells decreased obviously when GRP78 wasup-regulated by A23187, Which can be reversed by GRP78 knockdown after A23187induction.
5. It was obviously that increase of P53, P21 and P-CDC2 expression tendency duringcisplatin induction was slow down by GRP78 up-regulation.
6. The calcium concentration of kytoplasm increased after24h' cisplatin induction. But ifGRP78 was up-regulated by inductor, there was no change of hytoplasm calciumconcentration after24h' cisplatin induction. If GRP78 was knocked down afterup-regulation, the calcium concentration of kytoplasm can increase again with cisplatin.
7. For immunohistochemistry results, GRP78 expressed highly in normal cervical tissuesand precancerous change and cancer in situ, but express lowly in cancer tissues. GRP78expression was higher in ovarian cancer than in normal and benign tissues. GRP78expression had high dependability with HP1-γexpression in these two cancers.
Conclusion
1. As a new discovered chemotherapy induced-senescence associated regulating gene,GRP78 has rivalry and promotion with DNA damage-repair genes such as P53, P21 andCdc2. Down-regulation of GRP78 could promote the senescence induced by cisplatin.Nonetheless, up-regulation of GRP78 can decrease sensitivity of cisplatin-inducedsenescence. The phenomenon demonstrated that GRP78 may play a resistant role insenescence induced by cisplatin.
2. GRP78 may affect senescence sensitivity of tumour cells induced by cisplatin throughinfluencing DNA damage-repair pathway associated genes and change of kyroplasmcalcium concentration. GRP78 expression was higher in cervical precancerosis thanthat in normal tissues and cancer tissues. Moreover, GRP78 expression was highly relevant to HP1-γexpression, which meant that GRP78 may participate in thetransforming process from normal cells to malignant cells. GRP78 highly expressed inovarian cancer, which meant that it may influence chemotherapy effect of ovariancancer through anti-senescence.
Background and objective
In 1961, Hayflick discovered that there was limited proliferating capability in culturedcell in vitro. He named it as cell senescence. Tumor cells showed to be immortalized andcan proliferate unlimitedly. So tumor cell were considered to have a certain senescenceblock.. Nonetheless, recent studies established that tumor cells can be induced to besenescent under some conditions. For example, lower than apoptosis-induced dose DNAdamage agent can induce cell to have accelerated senescence, which was also calledpremature senescence. Other studies showed that senescence was an important mechanismprotecting normal cell from malignant transforming.
Esophageal carcinoma is one of the most common malignant tumor severily harmingpeople's life in our country. Esophageal carcinoma has a worse progonosis, nonetheless,which genes participate in its carcinogenesis and progression is still unknown. What rolethe senescence mechanism played in this process is also unclear. GRP78 gene was found tobe closely associated with senescence associated genes such as P53, which was discoveredas a new senescence regulating gene in our previous work. As one of the transcriptioninhibition factors, Heterochro-Matin-associated protein 1(HP1) is a component of thepolyprotein complex which could give rise to gene silencing. As a mark of senescenceassociated heterochromatin foci, HP1-γhas been widely used to show the cell and tissuesenescence. Owing to accessing paraffin section subjectively by staining intensity or area,the analysis results of traditional scoring method under light microscopy is lack of objectivity, which representativeness is not strong. Laser Scanning Cytometer(LSC)analyzes cells and morphous by sets of parameters, therefore it has been not only mainlyapplied to scan intracelluar materials and tissue quantitively, but also analyze the samepoint at different period of time. Because it can scan the selected region of entire paraffinsection at one time and can export objective and accurate result rapidly, LSC has beengradually used to analyze the immunohistochemistry results of tissue microarray. Based onstudy background and previous work above, we selected a tissue microarray containingdifferent grades of esophageal carcinoma and detected the expression of GRP78 and HP1-γprotein by immunohistochemistry. The results of immunohistochemistry were analyzedquantitatively by using laser scanning cytometer. We initially approach GRP78 andHP1-γexpression during malignant transformation of esophageal cell and their correlation.
Methods
A tissue microarray containing different grades of esophageal carcinoma wasselected. GRP78 and HP1-γprotein was detected by immunohistochemistry. The results ofimmunohistochemistry were analyzed quantitatively by laser scanning cytometer. Thecorrelation of GRP78 expression and HP1-γexpression was analyzed by chi square test.
Results
The phantoms and positive pixel percentage of selected areas were obtained afterscanning the entire tissue microarray. The positive rates of normal esophagus,moderate-severe atypical hyperplasia, carcinoma in site and squamous carcinoma were0%(0/8), 90.4%(19/21), 83.3%(5/6) and 47.4%(18/38), respectively. There was statisticaldiscrepancy of GRP78 expression among normal esophagus, moderate-severe atypicalhyperplasia plus carcinoma in site and squamous carcinoma(P＜0.01). HP1-γprotein wasprimarily expressed in the cell nucleus. The positive rate of normal esophagus,moderate-severe atypical hyperplasia, carcinoma in site and squamous cancer were 37.5%(3/8), 100% (21/21), 100%(6/6) and 23.7%(9/38), respectively. There was statistical discrepancy of HP1-γexpression among normal esophagus, moderate-severe atypicalhyperplasia plus carcinoma in site and squamous cancer(P＜0.01). There was highcorrelation between GRP78 expression and HP1-γexpression in esophageal carcinoma(P＜0.01).
Conclusion
There are many advantages such as high resolution, high objectivity and accuracywhen using laser scanning cytometer to analyze the immunohistochemistry results.Senescence may be an important mechanism for prevent normal cells from malignanttransformation. GRP78 highly expressed in precancerosis of esophageal and its expressionwas extremely correlated with HP-γexpression. These results demonstrate that GRP78 mayplay an important role in protecting normal cells from malignant transformation withsenescence.
引文
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3. Li WW, Sistonen L, Morimoto RL, Lee AS. Stress induction of the mammalian GRP78/Bip protein gene: in vivo genomic foot-printing and identification of p70 core from human nuclear extract as a DNA2binding component specific to the stress regulatory element. [J]. Mol Cell Biol, 1994,14 (8) :5533-46
4. Ng DT, Watowich SS, Lamb RA. Analysis in vivo of GRP78-Bip/ substrate interactions and their role in induction of the GRP78-Bip gene. [J]. Mol Biol Cell, 1992,3 (2) :143-55
5. Xu A, Bellamy AR, Taylor JA. Bip (GRP78) and endoplasmin (GRP94) are induced following rotavirus infection and bind transiently to an endoplasmic reticulum-localized virion component. [J]. J Virol, 1998, 72 (12) : 9865-72
6. Jorgensen MM, Jensen ON, Hoist HU et al. GRP78 is involved in retention of mutant low density lipoprotein receptor protein in the endoplasmic reticulum. [J]. J Biol Chem, 2000,275(43):33861-8
7. Miskovic D, Saiyer2Cid L , Ohan N et al . Isolation and characterization of a cDNA encoding a xenopus immunoglobulin binding protein, Bip (GRP78). [J]. Comp Biochem Physiol B Biochem Mol Biol, 1997 ,116 (2) :227-34
8. Techel D , Hafker T, Muschner S et al. Molecular analysis of a glucose-regulated gene (GRP78) of neurospora carssa. [J]. Biochem Bio phys Acta, 1998, 1397 (1) :21-26
9. Baqui MM, Gereben B , Harney JW et al . Distinct subcellular localization of transiently expresses type 1 and 2 iodothyronine deiodinasese as determined by immunofluorescence confocal microscopy. [J]. Endocri nology, 2000, 141 (11) : 4309-12
10. Mkrtchian S , Fang C , Hellman U , Ingelman Sundberg M. AStress-inducible ratliver endoplasmic reticulum protein, Erp29. [J]. Eur J Biochem, 1998, 251 (122): 304-13
11. Freiden PJ , Gaut JR , Hendershot LM. Interconversion of three differentially modified and assemble forms of Bip. [J]. EMBO J, 1992,11 (1): 63-70
12. Miyata T , Kokame K, Agarwala KL , Kato H. Analysis of gene expression in homocysteine injured vascular endothelial cells: Demonstration of GRP78/ Bip expression , cloning and characterization of a novel reducing agent-tunicamycin regulated gene. [J]. Semi nThromb Hemost, 1998, 24 (3): 285-91
13. Kokame K, Kato H , Miyats T. Homocysteine respondent genes in vascular endothelial cells identified by differential display analysis. GRP78/ Bip and novel genes. [J]. J Biol Chem, 1996, 271 (47): 29659-65
14. Liberman E, Fong YL, Selby MJ et al. Activation of the GRP78 and grp 94 promoters by hepatitis c virus E2 evelope protein. [J]. J Virol, 1999 ,73 (5) :3718-3722
15. Cai B , Tomida A , Mikami K, Nagata K Tsuruo T. Down-regulation of epidermal growth factor receptor signaling pathway by bing-ing of GRP78/ Bip to the receptor under glucose-starved stress conditions. [J]. J Cell Physiol, 1998,177 (2) :282-288
16. Lee AS. The glucose-regulated proteins : stress induction and clinical applications[J]. Trends iochem Sci, 2001, 26 (8): 504-510.
1. Hayrick L, Moorhead PS. The serial cultivation of human diploid cell strains. [J]. Exp Cell Res, 1961, 25: 585-621.
2. Miehaloglou C,Vredeveld LCW, SoengasMS, et al1BRAF (E600) 2asso2ciated senescence21ike cell cycle arrest of human naevi[J]Nature, 2005, 436 (7051) : 720-741
3. Linnik KM, Herscovitz H. Multiple molecular chaperone interact with apoliprotein B during its maturation: The network of endop lasmic reticulum2resident chaperones (Erp72, GRP94, calreticulin, and Bip interacts with apolip rotein b regardless of its lipidation state[J]. B iol Chem, 1998, 273 (33): 21368-21373.
4. Mkrtchian S, Fang C, Hellman U, et al. A stress2inducible ratliver endoplasmic retieulum protein, Erp29 [J]. Eur J B ioehem.1998, 251 (122): 304-313.
5. Mkrtehian S, Baryshev M, Matvijenko O, et al. Oligomerization properties of Erp29, an endoplasmic reticulum stress protein [J].FEBS Lett, 1998, 433 (3) : 335.
6. Narita M, Sabrina N, Heard E, et al. Rb-Mediated heterochromatin formation and silencing of E2F Target genes during cellular senescence. Cell, 2003, 113 (6): 703-716
7. Si JY, N ie SD, Chen Y, et al The development of image analyzing techno logy in immunoh istochernical micrograph.Foreign Medical Sciences (Biomedical Engineering Fascicle)，2002，25(3)：司京玉，聂生东，陈瑛，等．免疫组织化学显微图像分析技术进展．国外医学生物医学工程分册，2002，25(3)：117
1. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. [J]. Exp Cell Res, 1961, 25:585-621.
2. Itahana K, Camp isi J, Dimri GP. Methods to detect bio-markers of cellular senescence: the senescence-associated beta-galactosidase assay [J]. Methods Mol Biol, 2007, 371: 21 -31.
3. Narita M, Nunez S, Heard E, et al. Rb-mediated hetero-chromatin formation and silencing of E2F target genes during cellular senescence [J]. Cell, 2003, 113 (6) : 703-716.
4..Sharp less E, Chin L. The INK4a/ARF locus and melanoma [J]. Oncogene, 2003, 22 (20) : 3092-3098.
5. Collado M, Blasco MA, Serrano M. Cellular senescence in cancer and aging[J]. Cell, 2007, 130 (2):223-233.
6. Sarkisian CJ, KeisterBA, StairsDB, et al. Dose-dependent oncogene-induced senescence in vivo and its evasion duringmammary tumorigenesis [J]. Nat CellBiol, 2007, 9 (5): 493 - 505
7. Camp isi J. Suppressing cancer: the importance of being senescent [J]. Science, 2005, 309 (5736): 886 - 887.
8. KRTOLICA A , PARRINELLO S , LOCKETT S , et al. Senescent fibroblasts promote epithelial cell growth and tumorigenesis : a link between cancer and aging[J]. Proc Natl Acad Sci USA, 2001, 98 (21): 12072-12077.
9. D IMR I G P 1 What has senescence got to do with cancer [J] .Cancer Cell, 2005, 7 (6): 505-512
10. SerranoM, Lin AW, McCurrach ME, et al.Oncogenic ras p rovokes premature cell senescence associated with accumulation of p53 and pl6 ( INK4a) [J] Cell, 1997; 88 (5): 5932-6021
11. Bartkova J, Rezaei N, L iontos M, et al. Oncogene-induced senescence is part of the tumorigenesis barrier imposed byDNA damage checkpoints [J]. Nature, 2006, 444(7119): 633-637.
12. Bandyopadhyay D, Timchenko N, Suwa T, et al . The human melanocyte: a model system to study the complexity ofcellular aging and transformation in non fibroblastic cells [J]. Exp Gerontol, 2001, 36 ( 8) :1265-1275.
13. SAGE J, MILLER A, PEREZ2MANCERA P A, et all Acute mutation of retinoblastoma gene function is sufficient for cell cycle reentry[J]1 Nature, 2003, 424 (6945): 223-228
14. WEIW, HERB IG U, WEI S, et all Loss of retinoblastoma but not p16 function allows bypass of rep licative senescence in human fibroblasts[J].EMBO Rep, 2003, 4 (1) : 1061-1065
15. BEAUSEJOUR CM, KRTOL ICA A, GAL IM I F, et all Reversal of human cellular senescence: Roles of the p53 and pl6 pathways [J].EMBO J, 2003, 22 (16): 4212-4222
16. Fang X, Jin X, Xu HJ , et al. Expression of p16 induces transcriptional down regulation of the RB gene [J]. Oncogene, 1998, 16 (1):128.
17. Roux P. PTEN: a tumor suppressor with original properties [J].Bull Cancer ,1999 ,86 (6) :522 - 525.
18. Chen Z. Trotman LC, Shaffer D, et al. Crucial role of p53-dependent cellular senescence in supp ression of Pten deficient tumorigenesis [J]. Nature, 2005, 436 (7051):725-730.
19. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin2dependent kinases [J]. Genes Dev, 1995, 9 (10):1149-1163.
20. Palmero I, Mcconnell B, Parry D , et al . Accumulation of p16INK4a inmouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status [J]. Oncogene, 1997, 5 (5): 495-503.
21. Duan J, Zhang Z, Tong T. Senescence delay of human diploid fibroblastinduced by antisense p16INK4a expression [J]. J Biol Chem, 2001,276 (51) :48325-48331.
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24. Ressler, S., Bartkova, J., Niederegger, H., et al. p16INK4A is a robust in vivo biomarker of cellular aging in human skin. Aging Cell, 2006, 5:379-389.
25. Jacobs, J.J., Kieboom, K., Marino, S. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature, 1999, 397:164-168.
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1. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. [J]. Exp Cell Res, 1961, 25: 585-621.
2. Te Poele RH, et al. DNA damage is able to induce senescence in tumor cells in vitro and in vivo. [J]. Cancer Res, 2002, 62(6): 1876-1883
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1. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. [J]. Exp Cell R.es, 1961, 25:585-621.
2. Te Poele RH, et al. DNA damage is able to induce senescence in tumor cells in vitro and in vivo. [J]. Cancer Res, 2002, 62(6): 1876-1883
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1. Hayrick L, Moorhead PS. The serial cultivation of human diploid cell strains. [J]. Exp Cell Res, 1961, 25: 585-621.
2. Ortiz C, Cardemil L. Heat2shock responses in two leguminous plants: a comparative study [J]. Exp Bot, 2001, 52 (361): 1711-1719.
3. Li WW, Sistonen L, Morimoto RL, Lee AS. Stress induction of the mammalian GRP78/Bip protein gene: in vivo genomic foot-printing and identification of p70 core from human nuclear extract as a DNA2binding component specific to the stress regulatory element. [J]. Mol Cell Biol, 1994,14 (8) :5533-46
4. Ng DT, Watowich SS, Lamb RA. Analysis in vivo of GRP78-Bip/ substrate interactions and their role in induction of the GRP78-Bip gene. [J]. Mol Biol Cell, 1992,3 (2) :143-55
5. Xu A, Bellamy AR, Taylor JA. Bip (GRP78) and endoplasmin (GRP94) are induced following rotavirus infection and bind transiently to an endoplasmic reticulum-localized virion component. [J]. J Virol, 1998, 72 (12) : 9865-72
6. Jorgensen MM, Jensen ON, Hoist HU et al. GRP78 is involved in retention of mutant low density lipoprotein receptor protein in the endoplasmic reticulum. [J]. J Biol Chem, 2000,275(43):33861-8
7. Miskovic D, Saiyer2Cid L , Ohan N et al . Isolation and characterization of a cDNA encoding a xenopus immunoglobulin binding protein, Bip (GRP78). [J]. Comp Biochem Physiol B Biochem Mol Biol, 1997 ,116 (2) :227-34
8. Techel D , Hafker T, Muschner S et al. Molecular analysis of a glucose-regulated gene (GRP78) of neurospora carssa. [J]. Biochem Bio phys Acta, 1998, 1397 (1) :21-26
9. Baqui MM, Gereben B , Harney JW et al . Distinct subcellular localization of transiently expresses type 1 and 2 iodothyronine deiodinasese as determined by immunofluorescence confocal microscopy. [J]. Endocri nology, 2000, 141 (11) : 4309-12
10. Mkrtchian S , Fang C , Hellman U , Ingelman Sundberg M. AStress-inducible ratliver endoplasmic reticulum protein, Erp29. [J]. Eur J Biochem, 1998, 251 (122): 304-13
11. Freiden PJ , Gaut JR , Hendershot LM. Interconversion of three differentially modified and assemble forms of Bip. [J]. EMBO J, 1992,11 (1): 63-70
12. Miyata T , Kokame K, Agarwala KL , Kato H. Analysis of gene expression in homocysteine injured vascular endothelial cells: Demonstration of GRP78/ Bip expression , cloning and characterization of a novel reducing agent-tunicamycin regulated gene. [J]. Semi nThromb Hemost, 1998, 24 (3): 285-91
13. Kokame K, Kato H , Miyats T. Homocysteine respondent genes in vascular endothelial cells identified by differential display analysis. GRP78/ Bip and novel genes. [J]. J Biol Chem, 1996, 271 (47): 29659-65
14. Liberman E, Fong YL, Selby MJ et al. Activation of the GRP78 and grp 94 promoters by hepatitis c virus E2 evelope protein. [J]. J Virol, 1999 ,73 (5) :3718-3722
15. Cai B , Tomida A , Mikami K, Nagata K Tsuruo T. Down-regulation of epidermal growth factor receptor signaling pathway by bing-ing of GRP78/ Bip to the receptor under glucose-starved stress conditions. [J]. J Cell Physiol, 1998,177 (2) :282-288
16. Lee AS. The glucose-regulated proteins : stress induction and clinical applications[J]. Trends iochem Sci, 2001, 26 (8): 504-510.
1. Hayrick L, Moorhead PS. The serial cultivation of human diploid cell strains. [J]. Exp Cell Res, 1961, 25: 585-621.
2. Miehaloglou C,Vredeveld LCW, SoengasMS, et al1BRAF (E600) 2asso2ciated senescence21ike cell cycle arrest of human naevi[J]Nature, 2005, 436 (7051) : 720-741
3. Linnik KM, Herscovitz H. Multiple molecular chaperone interact with apoliprotein B during its maturation: The network of endop lasmic reticulum2resident chaperones (Erp72, GRP94, calreticulin, and Bip interacts with apolip rotein b regardless of its lipidation state[J]. B iol Chem, 1998, 273 (33): 21368-21373.
4. Mkrtchian S, Fang C, Hellman U, et al. A stress2inducible ratliver endoplasmic retieulum protein, Erp29 [J]. Eur J B ioehem.1998, 251 (122): 304-313.
5. Mkrtehian S, Baryshev M, Matvijenko O, et al. Oligomerization properties of Erp29, an endoplasmic reticulum stress protein [J].FEBS Lett, 1998, 433 (3) : 335.
6. Narita M, Sabrina N, Heard E, et al. Rb-Mediated heterochromatin formation and silencing of E2F Target genes during cellular senescence. Cell, 2003, 113 (6): 703-716
7. Si JY, N ie SD, Chen Y, et al The development of image analyzing techno logy in immunoh istochernical micrograph.Foreign Medical Sciences (Biomedical Engineering Fascicle)，2002，25(3)：司京玉，聂生东，陈瑛，等．免疫组织化学显微图像分析技术进展．国外医学生物医学工程分册，2002，25(3)：117
1. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. [J]. Exp Cell Res, 1961, 25:585-621.
2. Itahana K, Camp isi J, Dimri GP. Methods to detect bio-markers of cellular senescence: the senescence-associated beta-galactosidase assay [J]. Methods Mol Biol, 2007, 371: 21 -31.
3. Narita M, Nunez S, Heard E, et al. Rb-mediated hetero-chromatin formation and silencing of E2F target genes during cellular senescence [J]. Cell, 2003, 113 (6) : 703-716.
4..Sharp less E, Chin L. The INK4a/ARF locus and melanoma [J]. Oncogene, 2003, 22 (20) : 3092-3098.
5. Collado M, Blasco MA, Serrano M. Cellular senescence in cancer and aging[J]. Cell, 2007, 130 (2):223-233.
6. Sarkisian CJ, KeisterBA, StairsDB, et al. Dose-dependent oncogene-induced senescence in vivo and its evasion duringmammary tumorigenesis [J]. Nat CellBiol, 2007, 9 (5): 493 - 505
7. Camp isi J. Suppressing cancer: the importance of being senescent [J]. Science, 2005, 309 (5736): 886 - 887.
8. KRTOLICA A , PARRINELLO S , LOCKETT S , et al. Senescent fibroblasts promote epithelial cell growth and tumorigenesis : a link between cancer and aging[J]. Proc Natl Acad Sci USA, 2001, 98 (21): 12072-12077.
9. D IMR I G P 1 What has senescence got to do with cancer [J] .Cancer Cell, 2005, 7 (6): 505-512
10. SerranoM, Lin AW, McCurrach ME, et al.Oncogenic ras p rovokes premature cell senescence associated with accumulation of p53 and pl6 ( INK4a) [J] Cell, 1997; 88 (5): 5932-6021
11. Bartkova J, Rezaei N, L iontos M, et al. Oncogene-induced senescence is part of the tumorigenesis barrier imposed byDNA damage checkpoints [J]. Nature, 2006, 444(7119): 633-637.
12. Bandyopadhyay D, Timchenko N, Suwa T, et al . The human melanocyte: a model system to study the complexity ofcellular aging and transformation in non fibroblastic cells [J]. Exp Gerontol, 2001, 36 ( 8) :1265-1275.
13. SAGE J, MILLER A, PEREZ2MANCERA P A, et all Acute mutation of retinoblastoma gene function is sufficient for cell cycle reentry[J]1 Nature, 2003, 424 (6945): 223-228
14. WEIW, HERB IG U, WEI S, et all Loss of retinoblastoma but not p16 function allows bypass of rep licative senescence in human fibroblasts[J].EMBO Rep, 2003, 4 (1) : 1061-1065
15. BEAUSEJOUR CM, KRTOL ICA A, GAL IM I F, et all Reversal of human cellular senescence: Roles of the p53 and pl6 pathways [J].EMBO J, 2003, 22 (16): 4212-4222
16. Fang X, Jin X, Xu HJ , et al. Expression of p16 induces transcriptional down regulation of the RB gene [J]. Oncogene, 1998, 16 (1):128.
17. Roux P. PTEN: a tumor suppressor with original properties [J].Bull Cancer ,1999 ,86 (6) :522 - 525.
18. Chen Z. Trotman LC, Shaffer D, et al. Crucial role of p53-dependent cellular senescence in supp ression of Pten deficient tumorigenesis [J]. Nature, 2005, 436 (7051):725-730.
19. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin2dependent kinases [J]. Genes Dev, 1995, 9 (10):1149-1163.
20. Palmero I, Mcconnell B, Parry D , et al . Accumulation of p16INK4a inmouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status [J]. Oncogene, 1997, 5 (5): 495-503.
21. Duan J, Zhang Z, Tong T. Senescence delay of human diploid fibroblastinduced by antisense p16INK4a expression [J]. J Biol Chem, 2001,276 (51) :48325-48331.
22. OHTAN IN, ZEBEDEE Z, HUOT T J, et all Opposing effects of Ets and Id proteins on p16 INK4a exp ression during cellular senescence[J]. Nature, 2001, 409 (6823) : 1067-1070.
23. Krishnamurthy, J, Torrice, C, Ramsey, M.R., Kovalev, G,et al.. Ink4a/Arf expression is a biomarker of aging. J. Clin. Invest. 2004,114: 1299-1307.
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