【作者】 刘颜；

【导师】 王剑明；

【作者基本信息】 华中科技大学 ， 外科学， 2015， 博士

【摘要】 第一部分aPKC-τ和E-Cadherin在胆管癌组织中的表达及与预后的相关性研究目的：检测aPKC-τ和E-Cadherin在胆管癌组织中的表达,并探讨两者的表达与胆管癌患者临床预后的关系。方法：选取自2008年1月到2013年6月期间华中科技大学同济医学院附属同济医院胆胰外科64例经手术切除及经病理证实的胆管癌组织及10例胆总管囊肿病人胆管组织标本为研究对象。采用免疫组化(IHC)、实时定量聚合酶链反应(qRT-PCR)、免疫印迹法(Western blot)方法检测上述组织标本中aPKC-τ、E-Cadherin基因和蛋白的差异表达情况。采用卡方检验和t检验检测aPKC-τ和E-Cadherin的表达与临床病理分化和TNM分期的关系,采用Cox多因素回归分析胆管癌患者预后独立影响因素。结果：aPKC-τ在51.56%的胆管癌组织中呈高表达(33/64),在76.56%的癌旁组织中呈低表达(49/64),而正常组织中低表达则占100%(10/10)。aPKC-τ在胆管癌组织中的表达明显高于癌旁组织(x2=10.8,P=0.001)、正常组织(x2=9.306,P=0.002)。E-cadherin在67.2%的胆管癌组织中呈低表达(43/64),在32.8%的癌旁组织(21/64)和90%的正常组织(9/10)中呈高表达。E-cadherin在胆管癌组织中的表达明显低于癌旁组织(x2=16.949,P<0.001)、正常组织(x2=10.614,P<0.001)。aPKC-τ的表达水平与胆管癌分化程度呈负相关(x2=9.002,P==0.003),与胆管癌TNM分期密切相关,呈正相关(x2=7.000,P=0.008)。E-cadherin的表达水平与胆管癌分化程度呈正相关(x2=6.585,P<0.001),与胆管癌TNM分期呈负相关(x2=5.505,P<0.001)。Cox多因素回归分析显示,肿瘤分化程度、TNM分期、aPKC-i及E-cadherin表达水平是影响胆管癌患者预后的独立因素。结论：aPKC-i和E-Cadherin的表达水平与胆管癌的分化程度、TNM分期及预后密切相关,并且是胆管癌患者的独立预后因素。第二部分aPKC-τ通过Snail调节胆管癌细胞EMT目的：研究aPKC-τ调控胆管癌细胞EMT过程中的分子机制。方法：体外传代培养胆管癌细胞系TFK-1至对数期。合成携带aPKC-τ低表达(aPKC-i-siRNA)、aPKC-τ过表达(aPKC-τ-saRNA)的慢病毒包装质粒,并构建稳定aPKC-τ低表达TFK-1细胞(aPKC-τ-siRNA-TFK-1)、aPKC-、τ过表达TFK-1细胞(aPKC-τ-saRNA-TFK-1)、实验设置空白对照组(Blank)及阴性对照组(Control),并采用qRT-PCR、Western Blot检测不同实验组aPKC-τ、E-cadherin、Vimentin、Snai1、Snai2表达情况。为进一步检测aPKC-τ调节EMT过程中的关键作用靶点,选取aPKC-τ过表达组TFK-1(aPKC-τ-saRNA-TFK-1)细胞,运用Si-RNA技术靶向下调Snail表达,设置空白对照组(Blank),采用qRT-PCR、Western Blot检测不同实验组aPKC-τ、E-cadherin、Vimentin、Snail表达情况。结果：和Blank、Control组比较,伴随着aPKC-τ的表达下调,aPKC-τ-siRNA-TFK-1细胞中的E-cadherin表达量明显增高,Vimentin表达量则显著降低,Snail的表达量出现下降；而aPKC-τ过表达的aPKC-τ-saRNA-TFK-1细胞中上述标志物的表达情况则正好相反。各个实验组中Snai2的表达量则无明显变化。aPKC-i-saRNA-TFK-1细胞转染Si-Snail后,和对照组相比,实验组aPKC-τ表达无差异,伴随着Snail的表达量下降,其E-cadherin表达量出现升高,Vimentin表达量则出现下降。结论：aPKC-i是调节胆管癌EMT过程中的关键分子,并且该调节作用是依赖于转录因子Snail的介导作用来完成的。第三部分aPKC-τ调节胆管癌细胞化疗抵抗作用中的机制研究目的：研究aPKC-τ调控胆管癌细胞化疗抵抗作用中的机制。方法：体外传代培养胆管癌细胞系TFK-1至对数期。选取Blank、 aPKC-τ-siRNA-TFK-1、aPKC-i-saRNA-TFK-1细胞为实验对象,分别使用不同浓度吉西他滨(0.1μg/ml、1μg/ml、5μg/ml、10μg/ml)、顺铂(1μg/ml)、 10μg/ml、20μg/ml、50μg/ml)作用于上述细胞,采用CCK-8方法检测不同实验组细胞在药物作用12h、24h、48h、72h的细胞生存率,并计算不同实验组细胞的IC50值。采用流式细胞学方法检测不同实验组细胞在不同药物最佳浓度作用后细胞的凋亡情况。最后采用Western blot检测上述实验中药物抵抗蛋白P-gp、凋亡蛋白Caspase3的表达情况。结果：CCK-8检测结果显示,吉西他滨、顺铂对细胞的增殖抑制作用具有浓度及时间依赖性,随着药物作用浓度的增加和作用时间的延长,三种细胞的生存率均出现不同程度的下降。其中,aPKC-τ-siRNA-TFK-1细胞的下降程度最为明显。Blank、aPKC-τ-siRNA-TFK-1、aPKC-τ-saRNA-TFK-1细胞对吉西他滨的IC50值分别为1.52μg/ml、0.56μg/ml、2.78μg/ml(p<0.001),对顺铂的IC50值则分别为9.3μg/ml、5.7μg/ml、16.3μg/ml(P<0.001)。流式细胞学检测结果表明,药物对aPKC-τ-siRNA-TFK-1细胞的诱导凋亡作用最为明显,而aPKC-τ-saRNA-TFK-1细胞则未见明显凋亡。Western blot结果显示,aPKC-τ-siRNA-TFK-1细胞中药物抵抗蛋白P-gp的表达量较对照组明显下降,药物作用后凋亡蛋白Caspase3的表达较对照组明显升高；而aPKC-τ-saRNA-TFK-1细胞中的结果则相反。结论：aPKC-τ能够降低胆管癌细胞对化疗药物的敏感性,其作用机制可能是通过上调P-gp的表达来产生药物抵抗。同时,aPKC-i能通过下调凋亡蛋白Caspase3的表达,参与调节化疗药物诱导胆管癌细胞凋亡的过程。更多还原

【Abstract】 Part 1 Altered Expression of aPKC-i and E-Cadherin Correlate with Clinical Features in CholangiocarcinomaObjective:To investigate the correlations of aPKC-i and E-cadherin expression with clinicopathological characteristics, invasion, and metastasis, as well as with the prognosis of patients with cholangiocarcinoma (CCC).Methods:Carcinomatous and peritumoral tissues were obtained from 64 patients with CCC after resection without neoadjuvant, radiotherapy, or chemotherapy from January, 2008 to June,2013. And the normal tissues were obtained from 10 patients with Choledochalcyst. Both aPKC-τ and E-cadherin gene and protein expression were determined using reverse transcription polymerase chain reaction, immunohistochemistry, and western blotting. The relationship of aPKC-τ and E-cadherin expression with CCC clinical features was analyzed by Cox multivariate analysis. The data were analysed using the software package SPSS (version 19.0) for Windows supplied by the Statistics Teaching Room of Tongji Medical Collage, HUST. A value of P less than 0.05 was considered significant.Results:The expression of aPKC-τ was reduced in 100% of normal tissues (10/10) and 76.56% of peritumoral tissues (49/64), and increased aPKC-τ expression was detected in tumor tissues (33/64,51.56%). aPKC-τ expression were higher in tumor than in peritumoral (x2=10.8, P=0.001) and normal tissue (x2=9.306, P=0.002). In contrast, high E-cadherin expression was found in 90% of normal tissues (9/10) and 32.8% of peritumoral tissues (21/64), and E-cadherin expression was reduced in 67.2% of tumor tissues (43/64). E-cadherin expression were lower in tumor than in peritumoral (x2=16.949, P<0.001) and normal tissue (x2=10.614, P<0.001). Multivariate analysis showed tumor differentiation, TNM stage, aPKC-τ and E-cadherin were significant independent prognostic factors for overall survival in patients with CCC.Conclusion:This study demonstrates that abnormal expression of aPKC-τ and E-cadherin was closely related to tumor differentiation, TNM stage, as well as to patient survival.Part 2 aPKC-i regulates epithelial-mesenchymal transition in cholangiocarcinoma dependent on snailObjective:To study the molecular mechanisms of cholangiocarcinoma cells EMT process regulated by aPKC-τ.Methods:Cholangiocarcinoma cell line TFK-1 was cultured in vitro,and divided into 4 groups: Blank control、Negative control、aPKC-i-Si-RNA-TFK-1 and aPKC-i-SaRNA-TFK-1. aPKC-τ、E-cadherin、Vimentin、Snail and Snai2 were detected by Western Blot, the gene were detected by qRT-PCR. Then, we transfected Si-Snail to the aPKC-i-SaRNA-TFK-1, and detected the gene and protein of aPKC-τ、E-cadherin、 Vimentin、Snail.All data were expressed as the mean ±EM. Between group and among-group comparisons were conducted using Student t tests and ANOVA, respectively. Differences were considered statistically significant when P value was less than 0.05.Results:Cells overexpressing aPKC-τ became fibroblast-like in appearance and exhibited decreased levels of the epithelial marker E-cadherin but increased the expression levels of the mesenchymal markers Vimentin. Snail and Snai2 were the key transcription factors in EMT progression. Our data also showed that the expression of Snail was enhanced with aPKC-τ overexpression, whereas there was no significant change in Snai2 expression. In the aPKC-τ-Si-RNA-TFK-1 cell, Silencing of aPKC-τ induced an increase of E-cadherin,and a decrease of Vimentin, and Snail. Snai2 levels remained unaltered. To confirm the effects of Snail, we silenced Snail by transfecting Snail SiRNA in aPKC-i-SaRNA-TFK-1, and we found the downregulation of Snail reverses this process.Conclusion:In this study, we found that aPKC-τ plays an important role in the regulation of the process of cholangiocarcinoma cancer EMT, and the regulation dependent on Snail.Part 3 The mechanisms of chemotherapy resistance in cholangiocarcinoma regulated by aPKC-τObjective:To explore the chemotherapy resistance in cholangiocarcinoma cell of TFK-1 regulated by aPKC-τ.Methods:Detected the survival rates of cholangiocarcinoma cell lines TFK-1 treated with various doses of Gemcitabine or cisplatin by CCK-8.Detected the apoptosis rates of cholangiocarcinoma cell lines TFK-1 treated with various doses of Gemcitabine or cisplatin by flow cytometry. On the basis of the experimental results of flow and CCK-8, we test the protein expression of P-gp and Caspase3.Between group and among-group comparisons were conducted using Student t tests. Differences were considered statistically significant when P value was less than 0.05.Results:Under the concentration of 0.1μg/ml、lμg/ml、5μg/ml、10μg/ml Gemcitabine or 1μg/ml、10μg/ml、20μg/ml、50μg/ml Cisplatin treated, cell growth was inhibited and showed a dose-dependent manner. In Gemcitabine, the IC50 of Blank, aPKC-τ-siRNA-TFK-1, aPKC-t-saRNA-TFK-1 is 1.52ug/ml,0.56μg/ml,2.78μg/ml (P<0.001).In Cisplatin, the IC50 is 9.3μg/ml、5.7μg/ml、16.3μg/ml(P<0.001). After Gemcitabine(lμg/ml) or Cisplatin(10μg/ml) treated 48 hours, the apoptosis of aPKC-τ-siRNA-TFK-1 was higher than TFK-1, but the aPKC-τ-saRNA-TFK-1 was resistant to the apoptosis. Western blot analysis showed the expression of P-gp was the least in the aPKC-τ-siRNA-TFK-1, and the Caspase3 was the highest.Conclusion:Taken together, our results suggest that aPKC-t could regulated chemoresistance in cholangiocarcinoma cells dependent on upregulated P-gp. And, aPKC-τ could regulated apoptosis dependent on downregulated Caspase3.更多还原