自噬基因Beclin 1与宫颈癌相关性的实验研究
摘要
背景与研究目的
宫颈癌是女性生殖器官最常见的恶性肿瘤，全世界每年有50多万人患病，其中80％在发展中国家，严重威胁妇女的健康和生命。尽管宫颈癌的治疗取得了巨大进展，但晚期患者5年生存率仅为50％左右。生物治疗已成为目前肿瘤治疗的研究热点，但多以凋亡诱导为主，将自噬性细胞死亡引入肿瘤治疗，可能成为新的治疗靶点。
程序性细胞死亡可以分为凋亡和自噬两种方式，自噬作为Ⅱ型程序性细胞死亡，具有特殊的形态学改变和独有的调控通路，与凋亡截然不同。自噬是正常细胞的功能，是真核细胞蛋白降解的重要途径，是维持细胞自我平衡的重要机制，自噬异常与肿瘤形成有关。近来，随着人类自噬基因Beclin 1的克隆成功，进一步了解自噬在人类肿瘤中的作用，探索自噬调控通路，明晰自噬与凋亡的相互关系，成为目前的研究热点。
Beclin 1是新近克隆的哺乳动物的自噬调控基因，位于染色体17q21，在75％的卵巢癌，50％的乳腺癌和40％的前列腺癌中表现为单等位基因缺失，是候选的肿瘤抑制基因。关于Beclin 1与宫颈癌的相关性研究，目前尚未见到报道。本课题旨在研究自噬基因Beclin 1在宫颈癌组织中的表达，Beclin 1基因表达质粒及特异性RNA干扰质粒分别在体内、体外对宫颈癌HeLa细胞生长、凋亡、自噬的影响，探索自噬诱导在宫颈癌治疗中的可行性，为宫颈癌的基因治疗提供新的靶点。
方法通过免疫组化SP法检测Beclin 1蛋白在宫颈癌组织中的表达；通过目的基因克隆、载体构建，RNA干扰技术，构建Beclin 1基因表达质粒pcDNA3.1(+)—Beclin 1和RNA干扰质粒pSUPER—Beclin 1，分别观察它们在体内、外对宫颈癌HeLa细胞生长、自噬和凋亡的影响：
1．采用免疫组化SP法检测Beclin 1基因在宫颈癌组织(n=81)、CIN组织(n=20)、正常宫颈组织(n=20)中的表达状况，分析Beclin 1与宫颈癌临床分期、病理分级、淋巴结转移等临床病理因素的相关性；
2．通过RT-PCR方法，从人正常卵巢组织中获得Beclin 1基因，构建Beclin 1基因的真核表达质粒pcDNA3.1(+)—Beclin 1；采用计算机软件设计针对Beclin 1的特异性RNA干扰序列，构建Beclin 1基因的小发夹状RNA(shRNA)真核表达质粒pSUPER—Beclin 1；
3．利用脂质体将重组质粒pcDNA3.1(+)—Beclin 1和pSUPER—Beclin 1转染HeLa细胞，筛选稳定表达株，以荧光定量RT-PCR检测转染前后Beclin 1及caspase-9 mRNA的变化，以western blot检测转染前后Beclin 1及caspase-9蛋白的变化；采用流式细胞仪检测细胞周期、凋亡、自噬的变化；电镜检测自噬泡的形成，MDC染色检测自噬体的数量；MTT法检测细胞的增殖变化；
4．将上述两株稳定表达株分别接种到裸鼠皮下成瘤，建立雌性裸鼠异体移植宫颈癌模型，观察瘤体生长速度及瘤体大小，免疫组化检测瘤体组织中Beclin 1蛋白的表达。
采用，检验和x~2检验进行统计学分析。
结果
1．宫颈癌组织中Beclin 1的蛋白表达阳性率低于正常宫颈和CIN组织(P＜0.05)，宫颈癌中常见Beclin 1表达下调和缺失；正常宫颈与CIN间Beclin 1的表达无显著性差异(P＞0.05)；
2．Beclin 1表达与宫颈癌的临床分期、病理类型、宫颈浸润深度、宫颈肿瘤大小、肿瘤大体类型均无相关性(P＞0.05)；在高中分化组中Beclin 1的蛋白表达率高于低分化组，在有淋巴结转移组表达率低于无淋巴结转组(P＜0.05)；新辅助化疗前后Beclin 1蛋白的表达无显著性差异(P＞0.05)：
3．利用PCR分析，酶切鉴定及DNA测序证实Beclin 1基因的表达质粒pcDNA3.1(+)—Beclin 1和RNA干扰质粒pSUPER—Beclin 1构建成功；
4．Beclin 1基因表达质粒pcDNA3.1(+)—Beclin 1转染HeLa细胞后，Beclin 1、caspase-9 mRNA和蛋白的表达显著提高(P＜0.05)，与未转染组相比，G1期细胞比例明显增多，S期细胞比例明显降低，凋亡细胞和自噬细胞明显增加，并促进了自噬泡的形成，细胞生长活性降低；
5．Beclin 1基因的RNA干扰质粒pSUPER—Beclin 1转染HeLa细胞后，Beclin 1、caspase-9 mRNA和蛋白的表达明显降低(P＜0.05)，与未转染组相比，G1期细胞比例明显减少，S期细胞比例明显增高，凋亡细胞和自噬细胞明显减少，自噬泡形成减少，细胞生长活性增强；
6．表达质粒pcDNA3.1(+)—Beclin 1使HeLa细胞裸鼠体内Beclin 1蛋白表达增加，成瘤时间延长，细胞生长减慢，瘤体体积及重量明显变小，抑瘤率为52.15％；RNA干扰质粒pSUPER-Beclin 1则抑制Beclinl蛋白表达，缩短成瘤时间，肿瘤体积和重量增大达40.32％。
结论自噬基因Beclin 1与宫颈癌的发生、发展有关，自噬基因Beclin 1可有效调控肿瘤细胞周期，抑制宫颈癌细胞的恶性增殖，提高肿瘤细胞的自噬和凋亡能力，部分逆转其恶性表型，同时提高细胞的自噬和凋亡能力可能成为宫颈癌基因治疗的新靶点。
Background & Objective Cervical cancer is regarded as one of the mostcommon cancers in the world, accounting for approximately 500 000 newcases per year, and almost 80% of cases occur in developing countries.Despite all therapeutic efforts, the five-year survival of the advanced-stagecases is almost 50%. Biochemotherapy is an important method in the therapyof carcinoma, and the inducement of apoptosis is its main means, whileautophagic cell death may provide a new target.
Programmed cell death (PCD) is an essential and highly orchestratedprocess that plays a major role in morphogenesis and tissue homeostasisduring development. Two major types of PCD have been distinguished:apoptosis and autophagy. Autophagy is the bulk degradation of proteins andorganelles, a process essential for cellular maintenance, cell viability,differentiation and development in mammals, and may be defective in tumourcells. Autophagy in mammalian cells is important both for cellularremodeling during differentiation and for the negative regulation of tumorcell growth.
The mammalian gene encoding Beclin 1, a novel Bcl-2-interactingprotein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers andovarian cancers. Beclin 1 is a mammalian autophagy gene that can inhibittumorigenesis and is a candidate tumor suppressor. In the study,immunohistochemistry was employed to determine the expression of Beclin1 and explored its clinical significance in cervical cell carcinoma. Weconstructed the eukaryotic expression vector pcDNA3.1(+)-Beclinl andshRNA expression vector pSUPER-Beclinl, transfected into HeLa cells, tofred the effect of Beclin 1 overexpression and downexpression on thegrowth, autophagy and apoptosis of cervical cancer cell line HeLa in vitroand vivo, and explore the feasibility of induce of autophagy in treatment ofcervical carcinoma.
Methods
1. The expression of Beclin 1 was detected with SP immunohistochemistryin specimens of cervical cell carcinoma(n=81), cervical intraeoithelialneoplasm (n=20), and normal cervix (n=20). Correlations of expressionof Beclin 1 gene to clinicopathologic factors of cervical carcinoma werestatistically analyzed.
2. The eukaryotic expression vector pcDNA3.1(+)-Beclinl and shRNAexpression vectors pSUPER-Beclinl were constructed.
3. The vectors were transfected into HeLa cells via lipofectamine. Theexpression levels of Beclin 1 and caspase-9 mRNA and protein weredetected by real-time RT-PCR, western blot analysis in transfected cells.The cell cycles, autophagy, apoptosis and cell proliferations of HeLacells were measured with flow cytometry and MTT method aftertransfection. The ultrastructural analysis was under the electron microscope, and the formation of autophagic vacuoles was measured byMDC dyeing.
4. Human cervical cancer HeLa cell was transfected by vectorpcDNA3.1-Beclinl and pSUPER-Beclinl, and was seededhypopercutaneously on nude mice. The in vivo carcinogenic and growthactivities of cancer cell were observed, and Beclinl protein expression intumor tissues was detected by immunohistochemisty.
Results
1. Beclinl protein expression in tumor cells were down-regulation incervical invasive carcinoma, respectively, significantly lower than thosein normal cervix, and cervical intraeoithelial neoplasm (P＜0.05), thereno significant difference between the normal cervix, and cervicalintraeoithelial neoplasm (P＞0.05).
2. Expression of Beclin 1 has no correlation with FIGO stage, age, depth ofcervical infiltration, tumor size, and gross type of cervical lesion(P＞0.05),and correlation with pelvic lymph nodes metastases and tumorhistological grade (P＜0.05). The neoadjuvant chemotherapy had noinfluence on the expression ofBeclin 1 (P＞0.05).
3. PCR analysis and DNA sequencing confirmed that the eukaryoticexpression vector pcDNA3.1 (+)-Beclin 1 and shRNA expression vectorspSUPER-Beclinl were constructed successfully.
4. The eukaryotic expression vector pcDNA3.1(+)-Beclinl significantlyimproved the expressin of mRNA and protein of Beclinl and caspase-9 inHeLa cells (P＜0.05), the ratios of G1 phase of HeLa cell were increasedand the ratios of S phase were decreased significantly, and the cell proliferations of HeLa cell were inhibited, while more apoptosis cells andmore autophagy cells were identified in these cells.
5. The shRNA expression vectors pSUPER-Beclinl significantly inhibitedthe expressin of mRNA and protein of Beclinl and caspase-9 in HeLacells (P＜0.05), the ratios of G1 phase of HeLa cell were decreased andthe ratios of S phase were increased significantly, and the cellproliferations of HeLa cell were improved, while less apoptosis cells andless autophagy cells were identified.
6. After transfected by vector pcDNA3.1(+)—Beclin 1, the carcinogenicactivity of HeLa cell was decreased in nude mice, the volume and theweigh of tumors were reduced, and Beclin 1 protein level in tumor tissueswas improved. After transfected by vector pSUPER--Beclin 1, thecarcinogenic activity of HeLa cell was increased in nude mice, thevolume and the weigh of tumors were improved, and Beclin 1 proteinlevel in tumor tissues was reduced.
Conclusions Autophagy and apoptosis are two types of programmed celldeath, autophagy gene Beclin 1 play an important role in these two types, anddefect of autophagy and apoptosis may be important in tumor genesis. Thelevels of Beclin 1 expression reduced in cervical invasive carcinoma tissues,and this may be related with the occurrence and development of cervicalcancer. Beclin 1 transfection can inhibit the growth of HeLa cells in vitro andvivo. Beclin 1 may play an important role in generation and development ofcervical cancers, and it might be one of the ideal strategies for gene therapyof cervical cancer to improve the ability of autophagy and apoptosissimultaneously.
宫颈癌是女性生殖器官最常见的恶性肿瘤，全世界每年有50多万人患病，其中80％在发展中国家，严重威胁妇女的健康和生命。尽管宫颈癌的治疗取得了巨大进展，但晚期患者5年生存率仅为50％左右。生物治疗已成为目前肿瘤治疗的研究热点，但多以凋亡诱导为主，将自噬性细胞死亡引入肿瘤治疗，可能成为新的治疗靶点。
程序性细胞死亡可以分为凋亡和自噬两种方式，自噬作为Ⅱ型程序性细胞死亡，具有特殊的形态学改变和独有的调控通路，与凋亡截然不同。自噬是正常细胞的功能，是真核细胞蛋白降解的重要途径，是维持细胞自我平衡的重要机制，自噬异常与肿瘤形成有关。近来，随着人类自噬基因Beclin 1的克隆成功，进一步了解自噬在人类肿瘤中的作用，探索自噬调控通路，明晰自噬与凋亡的相互关系，成为目前的研究热点。
Beclin 1是新近克隆的哺乳动物的自噬调控基因，位于染色体17q21，在75％的卵巢癌，50％的乳腺癌和40％的前列腺癌中表现为单等位基因缺失，是候选的肿瘤抑制基因。关于Beclin 1与宫颈癌的相关性研究，目前尚未见到报道。本课题旨在研究自噬基因Beclin 1在宫颈癌组织中的表达，Beclin 1基因表达质粒及特异性RNA干扰质粒分别在体内、体外对宫颈癌HeLa细胞生长、凋亡、自噬的影响，探索自噬诱导在宫颈癌治疗中的可行性，为宫颈癌的基因治疗提供新的靶点。
方法通过免疫组化SP法检测Beclin 1蛋白在宫颈癌组织中的表达；通过目的基因克隆、载体构建，RNA干扰技术，构建Beclin 1基因表达质粒pcDNA3.1(+)—Beclin 1和RNA干扰质粒pSUPER—Beclin 1，分别观察它们在体内、外对宫颈癌HeLa细胞生长、自噬和凋亡的影响：
1．采用免疫组化SP法检测Beclin 1基因在宫颈癌组织(n=81)、CIN组织(n=20)、正常宫颈组织(n=20)中的表达状况，分析Beclin 1与宫颈癌临床分期、病理分级、淋巴结转移等临床病理因素的相关性；
2．通过RT-PCR方法，从人正常卵巢组织中获得Beclin 1基因，构建Beclin 1基因的真核表达质粒pcDNA3.1(+)—Beclin 1；采用计算机软件设计针对Beclin 1的特异性RNA干扰序列，构建Beclin 1基因的小发夹状RNA(shRNA)真核表达质粒pSUPER—Beclin 1；
3．利用脂质体将重组质粒pcDNA3.1(+)—Beclin 1和pSUPER—Beclin 1转染HeLa细胞，筛选稳定表达株，以荧光定量RT-PCR检测转染前后Beclin 1及caspase-9 mRNA的变化，以western blot检测转染前后Beclin 1及caspase-9蛋白的变化；采用流式细胞仪检测细胞周期、凋亡、自噬的变化；电镜检测自噬泡的形成，MDC染色检测自噬体的数量；MTT法检测细胞的增殖变化；
4．将上述两株稳定表达株分别接种到裸鼠皮下成瘤，建立雌性裸鼠异体移植宫颈癌模型，观察瘤体生长速度及瘤体大小，免疫组化检测瘤体组织中Beclin 1蛋白的表达。
采用，检验和x~2检验进行统计学分析。
结果
1．宫颈癌组织中Beclin 1的蛋白表达阳性率低于正常宫颈和CIN组织(P＜0.05)，宫颈癌中常见Beclin 1表达下调和缺失；正常宫颈与CIN间Beclin 1的表达无显著性差异(P＞0.05)；
2．Beclin 1表达与宫颈癌的临床分期、病理类型、宫颈浸润深度、宫颈肿瘤大小、肿瘤大体类型均无相关性(P＞0.05)；在高中分化组中Beclin 1的蛋白表达率高于低分化组，在有淋巴结转移组表达率低于无淋巴结转组(P＜0.05)；新辅助化疗前后Beclin 1蛋白的表达无显著性差异(P＞0.05)：
3．利用PCR分析，酶切鉴定及DNA测序证实Beclin 1基因的表达质粒pcDNA3.1(+)—Beclin 1和RNA干扰质粒pSUPER—Beclin 1构建成功；
4．Beclin 1基因表达质粒pcDNA3.1(+)—Beclin 1转染HeLa细胞后，Beclin 1、caspase-9 mRNA和蛋白的表达显著提高(P＜0.05)，与未转染组相比，G1期细胞比例明显增多，S期细胞比例明显降低，凋亡细胞和自噬细胞明显增加，并促进了自噬泡的形成，细胞生长活性降低；
5．Beclin 1基因的RNA干扰质粒pSUPER—Beclin 1转染HeLa细胞后，Beclin 1、caspase-9 mRNA和蛋白的表达明显降低(P＜0.05)，与未转染组相比，G1期细胞比例明显减少，S期细胞比例明显增高，凋亡细胞和自噬细胞明显减少，自噬泡形成减少，细胞生长活性增强；
6．表达质粒pcDNA3.1(+)—Beclin 1使HeLa细胞裸鼠体内Beclin 1蛋白表达增加，成瘤时间延长，细胞生长减慢，瘤体体积及重量明显变小，抑瘤率为52.15％；RNA干扰质粒pSUPER-Beclin 1则抑制Beclinl蛋白表达，缩短成瘤时间，肿瘤体积和重量增大达40.32％。
结论自噬基因Beclin 1与宫颈癌的发生、发展有关，自噬基因Beclin 1可有效调控肿瘤细胞周期，抑制宫颈癌细胞的恶性增殖，提高肿瘤细胞的自噬和凋亡能力，部分逆转其恶性表型，同时提高细胞的自噬和凋亡能力可能成为宫颈癌基因治疗的新靶点。
Background & Objective Cervical cancer is regarded as one of the mostcommon cancers in the world, accounting for approximately 500 000 newcases per year, and almost 80% of cases occur in developing countries.Despite all therapeutic efforts, the five-year survival of the advanced-stagecases is almost 50%. Biochemotherapy is an important method in the therapyof carcinoma, and the inducement of apoptosis is its main means, whileautophagic cell death may provide a new target.
Programmed cell death (PCD) is an essential and highly orchestratedprocess that plays a major role in morphogenesis and tissue homeostasisduring development. Two major types of PCD have been distinguished:apoptosis and autophagy. Autophagy is the bulk degradation of proteins andorganelles, a process essential for cellular maintenance, cell viability,differentiation and development in mammals, and may be defective in tumourcells. Autophagy in mammalian cells is important both for cellularremodeling during differentiation and for the negative regulation of tumorcell growth.
The mammalian gene encoding Beclin 1, a novel Bcl-2-interactingprotein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers andovarian cancers. Beclin 1 is a mammalian autophagy gene that can inhibittumorigenesis and is a candidate tumor suppressor. In the study,immunohistochemistry was employed to determine the expression of Beclin1 and explored its clinical significance in cervical cell carcinoma. Weconstructed the eukaryotic expression vector pcDNA3.1(+)-Beclinl andshRNA expression vector pSUPER-Beclinl, transfected into HeLa cells, tofred the effect of Beclin 1 overexpression and downexpression on thegrowth, autophagy and apoptosis of cervical cancer cell line HeLa in vitroand vivo, and explore the feasibility of induce of autophagy in treatment ofcervical carcinoma.
Methods
1. The expression of Beclin 1 was detected with SP immunohistochemistryin specimens of cervical cell carcinoma(n=81), cervical intraeoithelialneoplasm (n=20), and normal cervix (n=20). Correlations of expressionof Beclin 1 gene to clinicopathologic factors of cervical carcinoma werestatistically analyzed.
2. The eukaryotic expression vector pcDNA3.1(+)-Beclinl and shRNAexpression vectors pSUPER-Beclinl were constructed.
3. The vectors were transfected into HeLa cells via lipofectamine. Theexpression levels of Beclin 1 and caspase-9 mRNA and protein weredetected by real-time RT-PCR, western blot analysis in transfected cells.The cell cycles, autophagy, apoptosis and cell proliferations of HeLacells were measured with flow cytometry and MTT method aftertransfection. The ultrastructural analysis was under the electron microscope, and the formation of autophagic vacuoles was measured byMDC dyeing.
4. Human cervical cancer HeLa cell was transfected by vectorpcDNA3.1-Beclinl and pSUPER-Beclinl, and was seededhypopercutaneously on nude mice. The in vivo carcinogenic and growthactivities of cancer cell were observed, and Beclinl protein expression intumor tissues was detected by immunohistochemisty.
Results
1. Beclinl protein expression in tumor cells were down-regulation incervical invasive carcinoma, respectively, significantly lower than thosein normal cervix, and cervical intraeoithelial neoplasm (P＜0.05), thereno significant difference between the normal cervix, and cervicalintraeoithelial neoplasm (P＞0.05).
2. Expression of Beclin 1 has no correlation with FIGO stage, age, depth ofcervical infiltration, tumor size, and gross type of cervical lesion(P＞0.05),and correlation with pelvic lymph nodes metastases and tumorhistological grade (P＜0.05). The neoadjuvant chemotherapy had noinfluence on the expression ofBeclin 1 (P＞0.05).
3. PCR analysis and DNA sequencing confirmed that the eukaryoticexpression vector pcDNA3.1 (+)-Beclin 1 and shRNA expression vectorspSUPER-Beclinl were constructed successfully.
4. The eukaryotic expression vector pcDNA3.1(+)-Beclinl significantlyimproved the expressin of mRNA and protein of Beclinl and caspase-9 inHeLa cells (P＜0.05), the ratios of G1 phase of HeLa cell were increasedand the ratios of S phase were decreased significantly, and the cell proliferations of HeLa cell were inhibited, while more apoptosis cells andmore autophagy cells were identified in these cells.
5. The shRNA expression vectors pSUPER-Beclinl significantly inhibitedthe expressin of mRNA and protein of Beclinl and caspase-9 in HeLacells (P＜0.05), the ratios of G1 phase of HeLa cell were decreased andthe ratios of S phase were increased significantly, and the cellproliferations of HeLa cell were improved, while less apoptosis cells andless autophagy cells were identified.
6. After transfected by vector pcDNA3.1(+)—Beclin 1, the carcinogenicactivity of HeLa cell was decreased in nude mice, the volume and theweigh of tumors were reduced, and Beclin 1 protein level in tumor tissueswas improved. After transfected by vector pSUPER--Beclin 1, thecarcinogenic activity of HeLa cell was increased in nude mice, thevolume and the weigh of tumors were improved, and Beclin 1 proteinlevel in tumor tissues was reduced.
Conclusions Autophagy and apoptosis are two types of programmed celldeath, autophagy gene Beclin 1 play an important role in these two types, anddefect of autophagy and apoptosis may be important in tumor genesis. Thelevels of Beclin 1 expression reduced in cervical invasive carcinoma tissues,and this may be related with the occurrence and development of cervicalcancer. Beclin 1 transfection can inhibit the growth of HeLa cells in vitro andvivo. Beclin 1 may play an important role in generation and development ofcervical cancers, and it might be one of the ideal strategies for gene therapyof cervical cancer to improve the ability of autophagy and apoptosissimultaneously.
引文
1. Herrero R. Epidemiology of cervical cancer [J]. J Nali Cancer Inst Monogr, 1996, 21: 1-6.
2．郎景和。子宫颈上皮内瘤变的诊断与资料[J]．中华妇产科杂志，2001，36(5)：261-263．
3. Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol, 2004; 36(12):2405-2419.
4. Klionsky DJ. Autophagy. Current Biology, 2005 ; 15(8):282-283.
5. Kanzawa T, Germano IM, Komata T et al. Role of autophagy in temozolomide induced cytotoxicity for malignant glioma cells.Cell Death D iffer, 2004; 11 (4):448-57.
6. Yanagisawa H, Miyashita T, Nakano Y, et al. Hspinl, a transmembrane protein interacting with Bcl-2 /Bcl-xL, induces a caspase-independent autophagic cell death. Cell Death Differ, 2003; 10(7):798-807.
7. Chau YP,Lin SY, Chen JH, et al. Endostain induces autophagic cell death in Eahy926 human endothelial cells. Histol Histopathol, 2003, 18(3):715-726.
8. Liang X H, Kleeman L K, Jiang H H, et al. Protection against fatal Sindbis virus encephalitis by Beclin, a novel Bcl-2-interaeting protein. J Virol, 1998, 72(11): 8586-8596.
9. Liang XH,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by Beclin 1.Nature, 1999; 402(6762):672.
10. Aita, V. M. et al. Cloning and genomic structure of beclin 1, a candidate tumor suppressor gene on chromosome 17q21. Genomics , 1999, 59-65.
11. Schindl M, Bachtiary B, Dreier B, et al,Impact of human papillomavirus infection on the expression of the KAI1 metastasis suppressor protein in invasive cervical cancer.Cancer Lett. 2001,162(2):261-6.
12. Schindl M, Bimer P, Bachtiary B ,et al,The impact of expression of the metastasis suppressor protein KAI1 on prognosis in invasive squamous cell cervical cancer.Anticancer Res. 2000;20(6B):4551-5.
13. Kihara A,Kabeya Y, Ohsumi Y,et al. Beclin-phosphatidylinositol 3-kinase complex function at the trans-Golgi network[J].EMBO. 2001; 2(4):330-335.
14．赵剑虹，万小云，谢幸，等。Beclin1、PTEN在子宫内膜癌组织中的表达及其意义．癌症，2006；25(6)：753-757．
15. Trattner M, Graf AH, Lax S,et al.Prognostic factors in surgically treated stage Ⅰb-Ⅱb cervical carcinoma with special emphasis on the importance of tumor volume.Gynecol Oncol,2001,82(1): 11-16.
16. Kristen CB,Abeler VM,Risberg B,et al,Tumor size,depth of invasion, and grading of the invasive tumor front are the main prognostic factors in early squamous cell cervical careinoma.Gynecol Oncol, 1999, 74(2): 245-251.
17．连利娟，主编。林巧稚妇科肿瘤学。北京：人民卫生出版社，第三版，2000．273
18. Gorski D J, Chittaranjan S, Pleasance ED, et al. A SAGE approach to discovery of genes involved in autophagic cell death. Curr Biol, 2003; 13(4):358.
19. Lin HH,Cheng VF,Chan KW et al.Risk factor for recurrence in patients with stage Ⅰb,Ⅱa, and Ⅱb cervical carcinoma after radical hysterectony and post operative pelvic irradiation.Obstet Gynecol, 1996,88:274-279.
20. Sevin BU,Nadji M,Lampe B et al.Prognostic factors of early stage cervical cancer treated by radical hysterectomy.Cancer, 1995,76:1978-86 Cancer, 1995,64:355-359.
21. Duenas GA ,Lopez GC ,Gonzalez A ,et al. Induction chemotherapy with gemcitabine and oxaliplatin for locally advanced cervical carcinoma . Am J Clin Oncol, 2003; 26(1): 22-26.
22. Termrungruanglert W , Tresukosol D , Vasuratna A , et al. Neoadjuvant gemcitabine and cisplatin followed by radical surgery in ( bulky) squamous cell carcinoma of cervix stage Ib2. Gynecol Oncol, 2005; 97(2): 576-578.
23. Sambrook J, Russell DW 主编, 黄培堂等译. 分子克隆实验指南 (第三版) . 北京:科学出版社, 2002
24. Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature,2001; 411(24): 494-496.
25. I,age H. Potential applications of RNA inlerferenee technology in the treatulenl of cancer. Future Oncol, 2006; 1(1): 103-113.
26. Ketting RF, Fischer SE, Bernstein E. Dicer functions in RNA interfcrence anti in synthesis of small RNA involved in development timing in C. elegans. Genes Dev, 2001; 15(20): 2654-2659.
27. Pomerantz RJ. RNA intefie.fence meets HIV— 1: will silence be golden? Nat Med, 2002; 8(7): 659-660.
28. Yue Z, Jin S, Yang C, et al. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci USA, 2003; 100(25): 15077-15082.
29. Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest, 2003,112(12); 1809-1820.
30. Hoyer-Hansen M, Bastholm L, Mathiasen I S, et al. Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell Death Differ. 2005; 12(10); 1297-1309.
31. Biederbick A., Kern HF, Elsasser HP. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol, 1995; 66(1): 3-14.
32. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol, 2004; 36(12): 2491-2502.
33. Fan T, Han LH, Cong RS, et al. Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai), 2005; 37(11): 719-727.
34. Johnson CR, Jarvis WD. Caspase-9 regulation: an update[J]. Apoptosis, 2004; 9(4): 423-427.
35. Gorski DJ, Chittaranjan S, Pleasance ED, et al. A SAGE approach to discovery of genes involved in autophagic cell death. Curr Biol, 2003; 13(4):358-362.
36. Kanzawa T, Kondo Y, Ito H, et al. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res, 2003; 63(9):2103-2109.
37. YanagisawaH, MiyashitaT, Nakano Y, et al. HSpinl, a transmembrane protein interacting with Bcl-2/Bcl-xL, induces a caspase-independent autophagic cell death.Cell Death Differ,2003; 10(7):798-802.
38. Furuya D, Tsuji N. Beclin 1 augmented cis-diamminedic-hloroplatinum induced apoptosis via enhancing caspase-9 activity. Experimental Cell Research, 2005; 307(1):26-40.
39. Pattingre S,Levine B. Bcl-2 inhibition of autophagy: a new route to cancer?. Cancer Research, 2006; 66(6):2885-2888.
1. Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol, 2004; 36(12):2405-2419.
2. Klionsky DJ. Autophagy. Current Biology, 2005 ; 15(8):282-283.
3. Yu L, Lenardo MJ, Baehreche EH. Autophagy and caspases: a new cell death program. Cell Cycle, 2004; 3(9):1124-1126.
4. Codogno P, Meijer AJ. Autophagy and signaling: their role in cell survival and cell death. Cell Death & Differentiation, 2005; 12 (2) :1509-1518.
5. Klionsky D J, Cregg J M, Dunn A , et al. A unified nomenclature foryeast autophagy-related genes. Developmental Cell, 2003; 2(5): 539-545.
6. Mizushima N, Sugita H, Yoshimori T, et al. A new p rotein conjugation system essential for autophagy. J Bio Chem, 1998; 273(51): : 33889-33892.
7. Kivisako T, Ichimura Y, OkadaH, et al. The reversible modification regulates the membrane-binding state of Apg8 /Aut7 essential for autophagy and the cytoplam to vacuole targeting pathway. J Cell Biol, 2000; 151 (2) : 263 - 375.
8. Mizushima N, Hara T. Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration? Autophagy, 2006; 2(4): 302-304.
9. Mizushima N, Yamamoto A, Hatano M, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. Journal of Cell Biology, 2001: 152(4): 657-668.
10. Mizushima N, Kuma A, Kobayashi Y, et al. Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apgl2-Apg5 conjugate. Journal of Cell Science, 2003; 116(Pt9): 1679-1688.
11. Dzhagalov I, Chuck M, Mizushima N, et al. A critical role for the autophagy gene Atg5 in T cell survival and proliferation. Journal of Experimental Medicine, 2007; 204(1): 25-31.
12. Hirsako K, Imasato H, Hirota Y, et al. 3-Methyladenine specifically inhibits retrograde of cation2independentmannose 6-phosphate / in-sulin-like growth factor II receptor from the early endosome to the TGN. Biophys Res Commun, 2004; 316 (3) : 845-852.
13. Li C, Capan E, Zhao Y, et al. Autophagy is induced in CD4+ T cells and important for the growth factor-withdrawal cell death. Journal of Immunology, 2006; 177(8): 5163-5168.
14. Kamada Y, Funakoshi T, Shintani T, et al. Tor-mediated induction of autophagy via an Apgl protein kinase complex. J Cell Biol, 2000; 150: 1507-1513.
15. Scott RC, Schuldiner O, Neufeld TP. Role and regulation of starvation-induced autophagy in the Drosophila fat body.Dev Cell,2004; 7:167-178.
16. Christie GR, Hajduch E, Hundal HS, et al. Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-dependent manner. Journal of Biological Chemistry, 2002; 277(12): 9952-9957.
17. Arico S, Petiot A, Bauvy C, et al. The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidy-linositol 3-kinase/protein kinase B pathway. J Biol Chem, 2001; 276: 35243-35246.
18. Kihara A,Kabeya Y, Ohsumi Y,et al. Beclin-phosphatidylinositol 3-kinase complex function at the trans-Golgi network.EMBO, 2001; 2(4):330-335.
19. Liang XH, Jackson S, Seaman M, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature, 1999; 402:672-676.
20. Takeuchi H, Kanzawa T, Kondo Y, et al . Inhibition of platelet-derived growth factor signaling induced autophagy in maglignant glioma cell. BrJ Cancer, 2004; 90 (5): 1069-75.
21. Pattingre S , Bauvy C , Codogno P. Amino acids interfere with the ERK1/2-dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells. J Biol Chem, 2003; 278(19): 16667-16674.
22. Inbal B, Bialik S, Sabanay I, et al. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death.J Cell Biol, 2002; 157(3): 455-468.
23. Jang CW, Chen CH, Chen CC, et al. TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase. Nature Cell Biology, 2002; 4(1): 51-58.
24. Toth S, Nagy K, Palfia Z, Rez G. Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Cell Tissue Res, 2002; 309: 409-416.
25. Kanzawa T, Germano IM, Komata T et al. Role of autophagy in temozolomide induced cytotoxicity for malignant glioma cells. Cell Death Differ, 2004; 11 (4) : 448 - 457.
26. Komata T, Kanzawa T, Takeuchi H, el al. Antitumor effect of cyclin dependent kinase inhibitors p16 ( NK4A), p18 ( NK4C), p19(NK4D), p21 (WAF1 /CIP1 ) and p27 ( KIP1 ) on malignant glioma cells. B r J Cancer, 2003; 88 (8): 1277 -1280.
27. YanagisawaH, MiyashitaT, Nakano Y, et al. Hspinl, a transmembrane protein interacting with Bcl-2 /Bcl-xL, induces a caspase-independent autophagic cell death]. Cell Death Differ,2003; 10 (7): 798 - 807.
28. McCarthy NJ, Whyte MK, Gilbert CS, et al. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. Journal of Cell Biology, 1997; 136(1): 215-227.
29. Tsuneoka M, Umata T, Kimura H, et al. c-myc induces autophagy in rat 3Y1 fibroblast cells. Cell Structure & Function, 2003; 28(3): 195-204.
30. Kitanaka C , Kato K , Ijiri R , et al. Increased Ras expression and caspase-independent neuroblastoma cell death: possible mechanism of spontaneous neuroblastoma regression. Journal of the National Cancer Institute; 2002: 94(5): 358-368.
31. Pattingre S , Bauvy C , Codogno P. Amino acids interfere with the ERK1/2-dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells. Journal of Biological Chemistry, 2003; 278(19): 16667-16674.
32. Inoki K, Li Y, Zhu T, et al. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nature Cell Biology, 2002; 4(9): 648-657.
33. Liang XH,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by Beclin 1.Nature, 1999; 402(6762):672
34. Qu X, Yu J,Bhagat G et al. Promotion of tumorgenesis by heterozy gous disrup tion of the beclin 1 autopahgy gene [ J ]. J Clin Invest,2003; 112(12): 1809 -1820.
35. Liang XH, Yu J,Browm K, Levine B. Beclinl contains a leucine-rich nuclear export signal that is required for its autophagy and tumor supp ressor function. Cancer Res, 2001; 61(8): 3443-3449.
36. Toth S, Nagy K, Palfia Z, Rez G. Changes in cellular autophagic capacity during azaserine-initiated pancreatic carcinogenesis.Acta Biol Hung, 2001; 52 (4): 393 -401.
37. YaoKC, KomataT, Kondo Y, et al. Molecular response of human glioblastoma multiforme cells to ionizing radiation: cell cycle arrest, modulation of the expression of cyclin-dependent kinase inhibitors, and autophagy. Journal of Neurosurgery, 2003; 98(2): 378-384.
38. Chau YP, Lin SY, Chen JH, et al. Endostatin induces autophagic cell death in EAhy926 human endothelial cells. Histol Histopathol,2003; 18:715-726.
39. Wen S, Stolarov J, Myers MP, et al. PTEN controls tumor-induced angiogenesis. Proceedings of the National Academy of Sciences of the United States of America, 2001; 98(8): 4622-4627.
40. Bursch W, Ellinger A, Kienzl H., et al. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis, 1996; 17(8): 1595-1607.
41. Kanzawa T, Kondo Y, Ito H, et al. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Research, 2003; 63(9): 2103-2108.
42. Paglin S, Hollister T, Delohery T, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Research, 2001; 61(2): 439-444.
43. Butler R, Mitchell SH, TindallDJ, et al. Nonapoptotic cell death associated with S-phase arrest of prostate cancer cells via the peroxisome proliferator-activated receptor gamma ligand, 15-deoxy-delta12,14-prostaglandin J2. Cell Growth & Differentiation, 2000; 11(1): 49-61.
44. Gorski DJ, Chittaranjan S, Pleasance ED, et al. A SAGE approach to discovery of genes involved in autophagic cell death. Curr Biol, 2003; 13(4): 358-362.
45. Kanzawa T, Kondo Y, Ito H, et al. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res, 2003; 63(9): 2103-2109.
46. Yanagisawa H, Miyashita T, Nakano Y, et al. HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL, induces a caspase-independent autophagic cell death.Cell Death Differ, 2003; 10(7): 798-802.
47. Furuya D, Tsuji N. Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity. Experimental Cell Research, 2005; 307(1): 26-40.
48. Pattingre S, Levine B. Bcl-2 inhibition of autophagy: a new route to cancer?. Cancer Research, 2006; 66(6): 2885-2888.
1. Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol, 2004; 36(12):2405-2419.
2. Klionsky DJ. Autophagy. Current Biology, 2005 ; 15(8):282-283.
3. Codogno P, Meijer AJ. Autophagy and signaling: their role in cell survival and cell death. Cell Death & Differentiation, 2005; 12 (2) :1509-1518.
4. Yu L, Lenardo M J, Baehreche EH. Autophagy and caspases: a new cell death program. Cell Cycle, 2004; 3(9):1124-1126.
5. Codogno P, Meijer AJ. Autophagy and signaling: their role in cell survival and cell death. Cell Death & Differentiation, 2005; 12(2): 1509-1518.
6. Klionsky D J, Cregg J M, Dunn A , et al. A unified nomenclature foryeast autophagy-related genes. Developmental Cell, 2003; 2(5): 539-545.
7. Liang X H, Kleeman L K, Jiang H H, et al. Protection against fatal Sindbis virus encephalitis by Beclin, a novel Bcl-2-interacting protein. J Virol, 1998, 72(11): 8586-8596.
8. Liang XH,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by Beclin 1.Nature, 1999:402(6762):672
9. Aita, V. M. et al. Cloning and genomic structure of beclin 1, a candidate tumor suppressor gene on chromosome 17q21. Genomics , 1999, 59, 59-65
10．赵剑虹，万小云，谢幸，等．Beclin1、PTEN在子宫内膜癌组织中的表达及其意义．癌症，2006；25(6)：753-757．
11．王赞宏，彭芝兰，段振玲，等．自噬基因Beclin 1在宫颈鳞癌中的表达及其临床意义．四川大学学报(医学版)，2006；37(6)：860-863．
12. Yue Z, Jin S, Yang C, et al. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci USA, 2003,100(25): 15077-15082.
13. Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest, 2003,112(12): 1809-1820.
14. Hoyer-Hansen M, Bastholm L, Mathiasen I S, et al. Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell Death Differ. 2005,12(10): 1297-1309
15. Kihara A, Kabeya Y, Ohsumi Y, et al. Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep. 2001,2(4): 330-335.
16. Zeng X, Overmeyer JH, Maltese WAF.Unctional specificity of the mammalian Beclin-Vps34 PI3-kinase complex in macroautophagy versus endocytosis and lysosomal enzyme trafficking. EMBO Rep. 2001,2(4): 330-335
17. Liang X H,Yu J,Brown K, et al. Beclinl contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function.Cancer Res, 2001; 61 (8) : 3443-3449.
18. Gutierrez M, Master S, Singh S, et al. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell, 2004; 119(6): 753-66.
19. Yang G, Matsui Y, Sadoshima J. et al. Autophagy in chronically ischemic myocardium. Proceedings of the National Academy of Sciences of the United States of America, 2005; 102(39): 13807-13812.
20. Shibata M, Lu T, Furuya T, et al. Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1. Journal of Biological Chemistry, 2006; 281(20): 14474-14485.
21. Diskin T, Tal-Or P, Erlich S, et al. Closed head injury induces upregulation of Beclin 1 at the cortical site of injury. Journal of Neurotrauma, 2005; 22(7): 750-762.
22. Takacs-Vellai K, Vellai T, Puoti A, et al. Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans. Current Biology, 2005; 15(16): 1513-1517.
23. Boya P, Gonzalez-Polo RA, Casares N, et al. Inhibition of macroautophagy triggers apoptosis. Molecular & Cellular Biology, 2005; 25(3): 1025-1040.
24. Shimizu S, Kanaseki T, Mizushima N, et al. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nature Cell Biology, 2004; 6(12): 1221-1228.
25. Pattingre S, Tassa A, Qu X, et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell, 2005; 122(6): 927-939.
26. Pattingre S. Levine B. Bcl-2 inhibition of autophagy: a new route to cancer?. Cancer Research, 2006; 66(6): 2885-2888,.
27. Yu L, Alva A, Su H, et al. Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science, 2004; 304(5676): 1500-1502.
28. Furuya D , Tsuji N , Yagihashi A , et al. Beclin 1 augmented cis-diamminedichloropl-atinum induced apoptosis via enhancing caspase-9 activity. Experimental Cell Research, 2005; 307(1): 26-40.
29. Hoyer-Hansen M, Bastholm L, Mathiasen IS, et al. Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell Death & Differentiation, 2005; 12(10): 1297-1309.
30. Lamparska-Przybysz M, Gajkowska B, Motyl T. Cathepsins and BID are involved in the molecular switch between apoptosis and autophagy in breast cancer MCF-7 cells exposed to camptothecin. Journal of Physiology & Pharmacology, 2005; Suppl 3: 159-79.
31. Daniel F, Legrand A, Pessayre D, et al. Partial Beclin 1 silencing aggravates doxorubi-cin-and Fas-induced apoptosis in HepG2 cells. World Journal of Gastroenterology, 2006; 12(18): 2895-2900.
32. Scarlatti F, Bauvy C, Ventruti A, et al. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1. Journal of Biological Chemistry, 2004; 279(18): 18384-18391.
33. Lavieu G, Scarlatti , SalaG, et al. Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation. Journal of Biological Chemistry, 2006; 281(13): 8518-8527.
34. Reef S, Zalckvar E, Shifman O, et al. A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death. Molecular Cell, 2006; 22(4): 463-475.
35. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene, 2004; 23(16): 2891-2906.
2．郎景和。子宫颈上皮内瘤变的诊断与资料[J]．中华妇产科杂志，2001，36(5)：261-263．
3. Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol, 2004; 36(12):2405-2419.
4. Klionsky DJ. Autophagy. Current Biology, 2005 ; 15(8):282-283.
5. Kanzawa T, Germano IM, Komata T et al. Role of autophagy in temozolomide induced cytotoxicity for malignant glioma cells.Cell Death D iffer, 2004; 11 (4):448-57.
6. Yanagisawa H, Miyashita T, Nakano Y, et al. Hspinl, a transmembrane protein interacting with Bcl-2 /Bcl-xL, induces a caspase-independent autophagic cell death. Cell Death Differ, 2003; 10(7):798-807.
7. Chau YP,Lin SY, Chen JH, et al. Endostain induces autophagic cell death in Eahy926 human endothelial cells. Histol Histopathol, 2003, 18(3):715-726.
8. Liang X H, Kleeman L K, Jiang H H, et al. Protection against fatal Sindbis virus encephalitis by Beclin, a novel Bcl-2-interaeting protein. J Virol, 1998, 72(11): 8586-8596.
9. Liang XH,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by Beclin 1.Nature, 1999; 402(6762):672.
10. Aita, V. M. et al. Cloning and genomic structure of beclin 1, a candidate tumor suppressor gene on chromosome 17q21. Genomics , 1999, 59-65.
11. Schindl M, Bachtiary B, Dreier B, et al,Impact of human papillomavirus infection on the expression of the KAI1 metastasis suppressor protein in invasive cervical cancer.Cancer Lett. 2001,162(2):261-6.
12. Schindl M, Bimer P, Bachtiary B ,et al,The impact of expression of the metastasis suppressor protein KAI1 on prognosis in invasive squamous cell cervical cancer.Anticancer Res. 2000;20(6B):4551-5.
13. Kihara A,Kabeya Y, Ohsumi Y,et al. Beclin-phosphatidylinositol 3-kinase complex function at the trans-Golgi network[J].EMBO. 2001; 2(4):330-335.
14．赵剑虹，万小云，谢幸，等。Beclin1、PTEN在子宫内膜癌组织中的表达及其意义．癌症，2006；25(6)：753-757．
15. Trattner M, Graf AH, Lax S,et al.Prognostic factors in surgically treated stage Ⅰb-Ⅱb cervical carcinoma with special emphasis on the importance of tumor volume.Gynecol Oncol,2001,82(1): 11-16.
16. Kristen CB,Abeler VM,Risberg B,et al,Tumor size,depth of invasion, and grading of the invasive tumor front are the main prognostic factors in early squamous cell cervical careinoma.Gynecol Oncol, 1999, 74(2): 245-251.
17．连利娟，主编。林巧稚妇科肿瘤学。北京：人民卫生出版社，第三版，2000．273
18. Gorski D J, Chittaranjan S, Pleasance ED, et al. A SAGE approach to discovery of genes involved in autophagic cell death. Curr Biol, 2003; 13(4):358.
19. Lin HH,Cheng VF,Chan KW et al.Risk factor for recurrence in patients with stage Ⅰb,Ⅱa, and Ⅱb cervical carcinoma after radical hysterectony and post operative pelvic irradiation.Obstet Gynecol, 1996,88:274-279.
20. Sevin BU,Nadji M,Lampe B et al.Prognostic factors of early stage cervical cancer treated by radical hysterectomy.Cancer, 1995,76:1978-86 Cancer, 1995,64:355-359.
21. Duenas GA ,Lopez GC ,Gonzalez A ,et al. Induction chemotherapy with gemcitabine and oxaliplatin for locally advanced cervical carcinoma . Am J Clin Oncol, 2003; 26(1): 22-26.
22. Termrungruanglert W , Tresukosol D , Vasuratna A , et al. Neoadjuvant gemcitabine and cisplatin followed by radical surgery in ( bulky) squamous cell carcinoma of cervix stage Ib2. Gynecol Oncol, 2005; 97(2): 576-578.
23. Sambrook J, Russell DW 主编, 黄培堂等译. 分子克隆实验指南 (第三版) . 北京:科学出版社, 2002
24. Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature,2001; 411(24): 494-496.
25. I,age H. Potential applications of RNA inlerferenee technology in the treatulenl of cancer. Future Oncol, 2006; 1(1): 103-113.
26. Ketting RF, Fischer SE, Bernstein E. Dicer functions in RNA interfcrence anti in synthesis of small RNA involved in development timing in C. elegans. Genes Dev, 2001; 15(20): 2654-2659.
27. Pomerantz RJ. RNA intefie.fence meets HIV— 1: will silence be golden? Nat Med, 2002; 8(7): 659-660.
28. Yue Z, Jin S, Yang C, et al. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci USA, 2003; 100(25): 15077-15082.
29. Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest, 2003,112(12); 1809-1820.
30. Hoyer-Hansen M, Bastholm L, Mathiasen I S, et al. Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell Death Differ. 2005; 12(10); 1297-1309.
31. Biederbick A., Kern HF, Elsasser HP. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol, 1995; 66(1): 3-14.
32. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol, 2004; 36(12): 2491-2502.
33. Fan T, Han LH, Cong RS, et al. Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai), 2005; 37(11): 719-727.
34. Johnson CR, Jarvis WD. Caspase-9 regulation: an update[J]. Apoptosis, 2004; 9(4): 423-427.
35. Gorski DJ, Chittaranjan S, Pleasance ED, et al. A SAGE approach to discovery of genes involved in autophagic cell death. Curr Biol, 2003; 13(4):358-362.
36. Kanzawa T, Kondo Y, Ito H, et al. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res, 2003; 63(9):2103-2109.
37. YanagisawaH, MiyashitaT, Nakano Y, et al. HSpinl, a transmembrane protein interacting with Bcl-2/Bcl-xL, induces a caspase-independent autophagic cell death.Cell Death Differ,2003; 10(7):798-802.
38. Furuya D, Tsuji N. Beclin 1 augmented cis-diamminedic-hloroplatinum induced apoptosis via enhancing caspase-9 activity. Experimental Cell Research, 2005; 307(1):26-40.
39. Pattingre S,Levine B. Bcl-2 inhibition of autophagy: a new route to cancer?. Cancer Research, 2006; 66(6):2885-2888.
1. Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol, 2004; 36(12):2405-2419.
2. Klionsky DJ. Autophagy. Current Biology, 2005 ; 15(8):282-283.
3. Yu L, Lenardo MJ, Baehreche EH. Autophagy and caspases: a new cell death program. Cell Cycle, 2004; 3(9):1124-1126.
4. Codogno P, Meijer AJ. Autophagy and signaling: their role in cell survival and cell death. Cell Death & Differentiation, 2005; 12 (2) :1509-1518.
5. Klionsky D J, Cregg J M, Dunn A , et al. A unified nomenclature foryeast autophagy-related genes. Developmental Cell, 2003; 2(5): 539-545.
6. Mizushima N, Sugita H, Yoshimori T, et al. A new p rotein conjugation system essential for autophagy. J Bio Chem, 1998; 273(51): : 33889-33892.
7. Kivisako T, Ichimura Y, OkadaH, et al. The reversible modification regulates the membrane-binding state of Apg8 /Aut7 essential for autophagy and the cytoplam to vacuole targeting pathway. J Cell Biol, 2000; 151 (2) : 263 - 375.
8. Mizushima N, Hara T. Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration? Autophagy, 2006; 2(4): 302-304.
9. Mizushima N, Yamamoto A, Hatano M, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. Journal of Cell Biology, 2001: 152(4): 657-668.
10. Mizushima N, Kuma A, Kobayashi Y, et al. Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apgl2-Apg5 conjugate. Journal of Cell Science, 2003; 116(Pt9): 1679-1688.
11. Dzhagalov I, Chuck M, Mizushima N, et al. A critical role for the autophagy gene Atg5 in T cell survival and proliferation. Journal of Experimental Medicine, 2007; 204(1): 25-31.
12. Hirsako K, Imasato H, Hirota Y, et al. 3-Methyladenine specifically inhibits retrograde of cation2independentmannose 6-phosphate / in-sulin-like growth factor II receptor from the early endosome to the TGN. Biophys Res Commun, 2004; 316 (3) : 845-852.
13. Li C, Capan E, Zhao Y, et al. Autophagy is induced in CD4+ T cells and important for the growth factor-withdrawal cell death. Journal of Immunology, 2006; 177(8): 5163-5168.
14. Kamada Y, Funakoshi T, Shintani T, et al. Tor-mediated induction of autophagy via an Apgl protein kinase complex. J Cell Biol, 2000; 150: 1507-1513.
15. Scott RC, Schuldiner O, Neufeld TP. Role and regulation of starvation-induced autophagy in the Drosophila fat body.Dev Cell,2004; 7:167-178.
16. Christie GR, Hajduch E, Hundal HS, et al. Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-dependent manner. Journal of Biological Chemistry, 2002; 277(12): 9952-9957.
17. Arico S, Petiot A, Bauvy C, et al. The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidy-linositol 3-kinase/protein kinase B pathway. J Biol Chem, 2001; 276: 35243-35246.
18. Kihara A,Kabeya Y, Ohsumi Y,et al. Beclin-phosphatidylinositol 3-kinase complex function at the trans-Golgi network.EMBO, 2001; 2(4):330-335.
19. Liang XH, Jackson S, Seaman M, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature, 1999; 402:672-676.
20. Takeuchi H, Kanzawa T, Kondo Y, et al . Inhibition of platelet-derived growth factor signaling induced autophagy in maglignant glioma cell. BrJ Cancer, 2004; 90 (5): 1069-75.
21. Pattingre S , Bauvy C , Codogno P. Amino acids interfere with the ERK1/2-dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells. J Biol Chem, 2003; 278(19): 16667-16674.
22. Inbal B, Bialik S, Sabanay I, et al. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death.J Cell Biol, 2002; 157(3): 455-468.
23. Jang CW, Chen CH, Chen CC, et al. TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase. Nature Cell Biology, 2002; 4(1): 51-58.
24. Toth S, Nagy K, Palfia Z, Rez G. Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Cell Tissue Res, 2002; 309: 409-416.
25. Kanzawa T, Germano IM, Komata T et al. Role of autophagy in temozolomide induced cytotoxicity for malignant glioma cells. Cell Death Differ, 2004; 11 (4) : 448 - 457.
26. Komata T, Kanzawa T, Takeuchi H, el al. Antitumor effect of cyclin dependent kinase inhibitors p16 ( NK4A), p18 ( NK4C), p19(NK4D), p21 (WAF1 /CIP1 ) and p27 ( KIP1 ) on malignant glioma cells. B r J Cancer, 2003; 88 (8): 1277 -1280.
27. YanagisawaH, MiyashitaT, Nakano Y, et al. Hspinl, a transmembrane protein interacting with Bcl-2 /Bcl-xL, induces a caspase-independent autophagic cell death]. Cell Death Differ,2003; 10 (7): 798 - 807.
28. McCarthy NJ, Whyte MK, Gilbert CS, et al. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. Journal of Cell Biology, 1997; 136(1): 215-227.
29. Tsuneoka M, Umata T, Kimura H, et al. c-myc induces autophagy in rat 3Y1 fibroblast cells. Cell Structure & Function, 2003; 28(3): 195-204.
30. Kitanaka C , Kato K , Ijiri R , et al. Increased Ras expression and caspase-independent neuroblastoma cell death: possible mechanism of spontaneous neuroblastoma regression. Journal of the National Cancer Institute; 2002: 94(5): 358-368.
31. Pattingre S , Bauvy C , Codogno P. Amino acids interfere with the ERK1/2-dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells. Journal of Biological Chemistry, 2003; 278(19): 16667-16674.
32. Inoki K, Li Y, Zhu T, et al. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nature Cell Biology, 2002; 4(9): 648-657.
33. Liang XH,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by Beclin 1.Nature, 1999; 402(6762):672
34. Qu X, Yu J,Bhagat G et al. Promotion of tumorgenesis by heterozy gous disrup tion of the beclin 1 autopahgy gene [ J ]. J Clin Invest,2003; 112(12): 1809 -1820.
35. Liang XH, Yu J,Browm K, Levine B. Beclinl contains a leucine-rich nuclear export signal that is required for its autophagy and tumor supp ressor function. Cancer Res, 2001; 61(8): 3443-3449.
36. Toth S, Nagy K, Palfia Z, Rez G. Changes in cellular autophagic capacity during azaserine-initiated pancreatic carcinogenesis.Acta Biol Hung, 2001; 52 (4): 393 -401.
37. YaoKC, KomataT, Kondo Y, et al. Molecular response of human glioblastoma multiforme cells to ionizing radiation: cell cycle arrest, modulation of the expression of cyclin-dependent kinase inhibitors, and autophagy. Journal of Neurosurgery, 2003; 98(2): 378-384.
38. Chau YP, Lin SY, Chen JH, et al. Endostatin induces autophagic cell death in EAhy926 human endothelial cells. Histol Histopathol,2003; 18:715-726.
39. Wen S, Stolarov J, Myers MP, et al. PTEN controls tumor-induced angiogenesis. Proceedings of the National Academy of Sciences of the United States of America, 2001; 98(8): 4622-4627.
40. Bursch W, Ellinger A, Kienzl H., et al. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis, 1996; 17(8): 1595-1607.
41. Kanzawa T, Kondo Y, Ito H, et al. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Research, 2003; 63(9): 2103-2108.
42. Paglin S, Hollister T, Delohery T, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Research, 2001; 61(2): 439-444.
43. Butler R, Mitchell SH, TindallDJ, et al. Nonapoptotic cell death associated with S-phase arrest of prostate cancer cells via the peroxisome proliferator-activated receptor gamma ligand, 15-deoxy-delta12,14-prostaglandin J2. Cell Growth & Differentiation, 2000; 11(1): 49-61.
44. Gorski DJ, Chittaranjan S, Pleasance ED, et al. A SAGE approach to discovery of genes involved in autophagic cell death. Curr Biol, 2003; 13(4): 358-362.
45. Kanzawa T, Kondo Y, Ito H, et al. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res, 2003; 63(9): 2103-2109.
46. Yanagisawa H, Miyashita T, Nakano Y, et al. HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL, induces a caspase-independent autophagic cell death.Cell Death Differ, 2003; 10(7): 798-802.
47. Furuya D, Tsuji N. Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity. Experimental Cell Research, 2005; 307(1): 26-40.
48. Pattingre S, Levine B. Bcl-2 inhibition of autophagy: a new route to cancer?. Cancer Research, 2006; 66(6): 2885-2888.
1. Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol, 2004; 36(12):2405-2419.
2. Klionsky DJ. Autophagy. Current Biology, 2005 ; 15(8):282-283.
3. Codogno P, Meijer AJ. Autophagy and signaling: their role in cell survival and cell death. Cell Death & Differentiation, 2005; 12 (2) :1509-1518.
4. Yu L, Lenardo M J, Baehreche EH. Autophagy and caspases: a new cell death program. Cell Cycle, 2004; 3(9):1124-1126.
5. Codogno P, Meijer AJ. Autophagy and signaling: their role in cell survival and cell death. Cell Death & Differentiation, 2005; 12(2): 1509-1518.
6. Klionsky D J, Cregg J M, Dunn A , et al. A unified nomenclature foryeast autophagy-related genes. Developmental Cell, 2003; 2(5): 539-545.
7. Liang X H, Kleeman L K, Jiang H H, et al. Protection against fatal Sindbis virus encephalitis by Beclin, a novel Bcl-2-interacting protein. J Virol, 1998, 72(11): 8586-8596.
8. Liang XH,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by Beclin 1.Nature, 1999:402(6762):672
9. Aita, V. M. et al. Cloning and genomic structure of beclin 1, a candidate tumor suppressor gene on chromosome 17q21. Genomics , 1999, 59, 59-65
10．赵剑虹，万小云，谢幸，等．Beclin1、PTEN在子宫内膜癌组织中的表达及其意义．癌症，2006；25(6)：753-757．
11．王赞宏，彭芝兰，段振玲，等．自噬基因Beclin 1在宫颈鳞癌中的表达及其临床意义．四川大学学报(医学版)，2006；37(6)：860-863．
12. Yue Z, Jin S, Yang C, et al. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci USA, 2003,100(25): 15077-15082.
13. Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest, 2003,112(12): 1809-1820.
14. Hoyer-Hansen M, Bastholm L, Mathiasen I S, et al. Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell Death Differ. 2005,12(10): 1297-1309
15. Kihara A, Kabeya Y, Ohsumi Y, et al. Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep. 2001,2(4): 330-335.
16. Zeng X, Overmeyer JH, Maltese WAF.Unctional specificity of the mammalian Beclin-Vps34 PI3-kinase complex in macroautophagy versus endocytosis and lysosomal enzyme trafficking. EMBO Rep. 2001,2(4): 330-335
17. Liang X H,Yu J,Brown K, et al. Beclinl contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function.Cancer Res, 2001; 61 (8) : 3443-3449.
18. Gutierrez M, Master S, Singh S, et al. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell, 2004; 119(6): 753-66.
19. Yang G, Matsui Y, Sadoshima J. et al. Autophagy in chronically ischemic myocardium. Proceedings of the National Academy of Sciences of the United States of America, 2005; 102(39): 13807-13812.
20. Shibata M, Lu T, Furuya T, et al. Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1. Journal of Biological Chemistry, 2006; 281(20): 14474-14485.
21. Diskin T, Tal-Or P, Erlich S, et al. Closed head injury induces upregulation of Beclin 1 at the cortical site of injury. Journal of Neurotrauma, 2005; 22(7): 750-762.
22. Takacs-Vellai K, Vellai T, Puoti A, et al. Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans. Current Biology, 2005; 15(16): 1513-1517.
23. Boya P, Gonzalez-Polo RA, Casares N, et al. Inhibition of macroautophagy triggers apoptosis. Molecular & Cellular Biology, 2005; 25(3): 1025-1040.
24. Shimizu S, Kanaseki T, Mizushima N, et al. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nature Cell Biology, 2004; 6(12): 1221-1228.
25. Pattingre S, Tassa A, Qu X, et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell, 2005; 122(6): 927-939.
26. Pattingre S. Levine B. Bcl-2 inhibition of autophagy: a new route to cancer?. Cancer Research, 2006; 66(6): 2885-2888,.
27. Yu L, Alva A, Su H, et al. Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science, 2004; 304(5676): 1500-1502.
28. Furuya D , Tsuji N , Yagihashi A , et al. Beclin 1 augmented cis-diamminedichloropl-atinum induced apoptosis via enhancing caspase-9 activity. Experimental Cell Research, 2005; 307(1): 26-40.
29. Hoyer-Hansen M, Bastholm L, Mathiasen IS, et al. Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell Death & Differentiation, 2005; 12(10): 1297-1309.
30. Lamparska-Przybysz M, Gajkowska B, Motyl T. Cathepsins and BID are involved in the molecular switch between apoptosis and autophagy in breast cancer MCF-7 cells exposed to camptothecin. Journal of Physiology & Pharmacology, 2005; Suppl 3: 159-79.
31. Daniel F, Legrand A, Pessayre D, et al. Partial Beclin 1 silencing aggravates doxorubi-cin-and Fas-induced apoptosis in HepG2 cells. World Journal of Gastroenterology, 2006; 12(18): 2895-2900.
32. Scarlatti F, Bauvy C, Ventruti A, et al. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1. Journal of Biological Chemistry, 2004; 279(18): 18384-18391.
33. Lavieu G, Scarlatti , SalaG, et al. Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation. Journal of Biological Chemistry, 2006; 281(13): 8518-8527.
34. Reef S, Zalckvar E, Shifman O, et al. A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death. Molecular Cell, 2006; 22(4): 463-475.
35. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene, 2004; 23(16): 2891-2906.
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