【作者】 刘东雷；

【导师】 王建军；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 第一部分：自噬相关蛋白在食管鳞癌中的表达及其临床意义目的已有研究表明细胞自噬水平的变化与肿瘤的发生、发展有关。本研究探讨自噬相关蛋白Beclin1和LC3食管鳞状细胞癌中的表达情况,并分析其与各种临床病理因素之间的相关性。方法51例食管鳞癌病例标本和30例癌旁正常食管组织均来自华中科技大学同济医学院附属协和医院病理科2008年3-12月在手术切除存档蜡块。采用免疫组织化学检测自噬相关蛋白Beclin1和LC3的表达,使用SPSS13.0软件进行统计学分析。结果Beclin1和LC3的阳性表达率分别为43.1%和39.3%。食管鳞癌组Beclin 1和LC3的表达明显低于癌旁组织(86.7%和83.3%),且差异具备有统计学意义(P<0.05)。Beclin 1和LC3的表达强度与肿瘤TNM分期和临床分期相关(P值均<0.05)；Beclin1与LC3在食管鳞癌中的表达呈正相关(P<0.01)。结论Beclin 1和LC3在食管鳞癌中的表达下调；自噬活性的下调可能与食管癌的发生、发展相关,并在食管癌的侵袭和转移中发挥重要作用。第二部分：顺铂诱导食管癌细胞EC9706自噬的研究目的检测顺铂对食管癌EC9706细胞增殖的抑制作用,观察在这一过程中细胞自噬水平的变化,并初步探讨影响白噬可能的机制。方法采用CCK-8法检测顺铂对EC9706细胞增殖的抑制作用,MDC荧光染色检测细胞内自噬囊泡的数量。Western blot法检测Beclin1和LC3蛋白的表达,RT-PCR检测顺铂对EC9706细胞Beclin1 mRNA的表达。结果顺铂对EC9706细胞有着显著的增殖抑制作用。顺铂处理EC9706细胞24h后,MDC染色显示DDP组细胞内出现明显的绿色荧光颗粒,说明顺铂能够诱导EC9706细胞产生自噬囊泡；而无论是mRNA还是蛋白水平,EC9706在顺铂处理后自噬水平明显提高,此变化一直持续到给药24h,且给药后6h表达最强,而对照组自噬改变不明显。结论顺铂对食管癌EC9706细胞有明显的生长抑制作用。顺铂可以诱导EC9706细胞发生自噬,此变化一直持续到给药24h,且给药后6h表达最强,其机制可能与Beclin1蛋白有关。第三部分：自噬在顺铂诱导食管癌细胞死亡中的作用目的观察顺铂联合自噬特异性抑制剂3-MA对EC9706增殖抑制的影响,探讨自噬在顺铂诱导的食管癌EC9706细胞死亡中的作用。方法CCK-8法测定顺铂联合3-MA对EC9706细胞增殖作用的影响；流式细胞仪检测3-MA联合顺铂作用EC9706细胞作用过程中细胞凋亡和细胞周期的改变；Western blot法检测顺铂对EC9706细胞PI3KⅢ和细胞色素c蛋白表达的影响。结果3-MA联合顺铂处理EC9706细胞24h后,MDC染色显示在加用3-MA组细胞内的绿色荧光颗粒与DDP单独处理组相比明显减少,提示这一自噬过程能够被自噬特异性抑制剂3-MA所抑制。CCK-8结果显示24h处理后DDP+3-MA组细胞死亡率较DDP组显著提高。流式细胞仪结果显示DDP+3-MA组细胞凋亡率较DDP单独处理组明显升高,并且DDP+3-MA组细胞周期中S期细胞的比例明显升高,同时伴随着G2/M期细胞的降低。Western blot结果显示与DDP组相比,DDP+3-MA组PI3KⅢ蛋白的表达受到了明显的抑制；cyt-c蛋白的释放明显增加；说明在顺铂诱导EC9706细胞自噬的过程中,自噬可能是通过Ⅲ型PI3K通路来实现的,而细胞凋亡主要是通过促进线粒体内cyt-c蛋白向细胞胞浆内释放来实现的。结论自噬特异性抑制剂3-MA能够通过抑制自噬提高顺铂对食管癌EC9706细胞的细胞毒性,其上游机制可能是激活Ⅲ型PI3K通路；而顺铂诱导EC9706细胞的凋亡下游主要是通过细胞内cyt-c的释放和将细胞周期阻滞在S期来实现,这说明抑制自噬可能是食管癌治疗的一个新的治疗方向。更多还原

【Abstract】 PART I:Expression and Clinical Significance of Autophagy-related Proteins in Esophageal Squamous Cell CarcinomaObjective:Many studies have indicated that the change of autophagy might be related with the growth and development of tumor. This study was to investigate the expression and implication of atuophagy-related gene Beclinl and microtubule-associated protein 1 light chain 3 (LC3) in esophageal squamous cell carcinoma (ESCC) and their relationships with clinical pathological features of ESCC.Methods:Immunohistochemistry was used to detect the expression of Beclin 1 and LC3 proteins in 51 ESCC tissues and 30 normal para-cancerous tissues. And the correlations of their expression to the clinicopathologic characterisics were analyzed by SPSS 13.0.Results:The positive rates of Beclinl and LC3 were 62.7%(32/51) and 66.7%(34/51) respectively. The expression of Beclinl and LC3 protein was detected in a significantly greater proportion in ESCC carcinoma tissues than that in para-cancerous tissues (15.7% and 9.80%, P<0.05). The positive rates of Beclinl and LC3 were correlated with lymph node metastasis and TNM stage. There was no relationship was between the two factors and age, sex, pathological subtypes and histological grades. Beclinl expression was associated with LC3 expression (P<0.05). Beclin 1 and LC3 were over-expressed in ESCC.Conclusion:LC3 and Beclinl expresson are up-regulated in ESCC. The increase of autophagic capacity may be involved in the carcinogenesis of ESCC, and play an important role in invasion and metastasis of ESCC. PartⅡ:Role of Autophagy in Esophageal Carcinoma Cell Line induced by CisplatinObjective:To explore the expression of autophagy in EC9706 cell line with treatment of Cisplatin, and study whether the expression level of autophagy is related to the death induced by Cisplatin.Methods:CCK-8 assays were used to detect the growth inhibition rate, and MDC staining was used to examine the autophagosome. Western bolt was used to detect the protein expression of Beclinl and LC3. RT-PCR analysis was used to dectect the expression of Beclin 1 mRNA.Results:DDP can evidently inhibit the growth of EC9706 cell line. After DDP treatment for 24h, MDC staining indicated that autophagosomes were increased in DDP group with respect to the control group, which showed that autophagy was induced by DDP in EC9706 cells. From the expression of LC3 and Beclinl by Western blot and the Beclin 1mRNA expression by RT-PCR, the perk of autphagy indcued by DDP is 6 hours and the expression of autophagy can continue for 24 hours. There was no obvious change the control group.Conclusion:Cisplatin can strongly inhibit proliferation of EC9706. DDP can induce autophagy in EC9706 cells. The expression of autophagy induced by DDP in EC9706 cells may be related with Beclinl signal transduction pathway. Part III:Role of Autophagy in esophageal carcinoma cell line induced by cisplatinObjective:To explore the role of autophagy in cisplatin-induced death of EC9706 cell line.Methods:To explore the role of autophagy in cisplatin-induced death of EC9706 cell line, the autophagy specific inhibitor 3-MA was added with cisplatin. The apoptosis rate and cell death rate were examined by flow cytometry and CCK-8 assays. Western bolt was used to detect the protein expression of PI3K III and cyt-c induced by cisplatin in EC9706 cell line.Results:After treatment of EC9706 cells with 3-MA and cisplatin for 24h, MDC staining indicated that autophagy induced by cisplatin was attenuated by 3-MA. course of treatment. Moreover, after the combination treatment of 3-MA and DDP, the cell inhibitory rate increased; the apoptosis rate and the numbers of cells in S phase also increased. Furthermore, the accumulation of autophagic vacuoles was decreased; the expression of Beclinl and LC3 was significantly down-regulated and the release of cytochrome c was decreased. DDP-induced apoptosis in EC9706 cells can be enhanced by the inhibitor of autophagy,3-MA.Conclusion:3-MA could attenuate cisplatin-induced autphagy of EC9706 cells and enhace the inhibitory effect on EC9706 cells. Cisplatin induced apoptosis and activated cyt-c in EC9706 cells. In a word, the inhibition of autophagy could be a novel strategy for the adjuvant chemotherapy of esophageal cancer更多还原