Salubrinal协同雷帕霉素抑制人胆管癌细胞的作用及其机制

发布时间：2018-06-07 03:34

本文选题：Salubrinal + 协同　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:研究真核翻译起始因子2α(eukaryotic translation initiation factors2,e IF2α)抑制剂salubrinal(sal)协同雷帕霉素(rapamycin,rap)对人胆管癌细胞的抑制效果及其分子机制。方法:培养人胆管癌细胞系QBC939和RBE细胞,用rap抑制胆管癌细胞m TOR信号通路,采用e IF2α抑制剂sal单独或与rap联合处理细胞、Bcl-x L抑制剂ABT-737单独或与rap联合处理细胞,通过向裸鼠皮下注射QBC939实现荷瘤,荷瘤后给不同实验组加相应的药物进行干预,监测小鼠荷瘤效果,用免疫印迹分析药物干预后肿瘤细胞和成瘤癌组织中相关分子以及凋亡相关分子PARP、Cleaved Caspase-3表达水平,用CCK-8检测细胞增殖速率,免疫组化分析药物干预对小鼠荷瘤组织增殖的影响。结果:(1)Rap抑制CCA细胞增殖:免疫印迹(Western blot)分析结果说明rap可以抑制CCA细胞中m TOR通路的活化,CCK-8检测证实rap对QBC939和RBE细胞的增殖起抑制作用,并且呈时间依赖性抑制(P0.05);此外,rap未引起QBC939和RBE细胞明显凋亡(数据未展示);(2)Sal抑制CCA细胞增殖:Western blot结果表明e IF2α/ATF4信号通路在CCA细胞中呈过度活化状态,并且sal可以抑制e IF2α的活性,CCK-8检测证实sal对QBC939和RBE细胞的增殖起抑制作用,并且呈时间依赖性抑制(P0.05);此外,sal未引起QBC939和RBE细胞明显凋亡(数据未展示);(3)Sal抑制CCA细胞成瘤:在体内实验中,sal抑制QBC939在裸鼠体内的荷瘤能力,并通过免疫组化证实sal抑制QBC939细胞体内细胞增殖能力;(4)Sal和rap联合应用抑制CCA细胞成瘤:在裸鼠体内sal和rap单独干预抑制QBC939细胞的成瘤,二者联合干预协同抑制QBC939细胞的成瘤;(5)Sal和rap联合应用协同抑制CCA细胞成瘤的机制:免疫组化证实sal和rap联合干预比单独sal或rap干预表现出对荷瘤的QBC939细胞生长更强的抑制作用;在体外实验中,CCK-8分析结果提示sal和rap对QBC939细胞增殖有协同抑制作用(P0.05)。用Western blot分析表明在QBC939细胞中sal处理不仅可以下调p-AKT的表达还可消除rap诱导的p-AKT表达上调;在体外实验中,Western blot结果表明sal和rap单独或联合应用未引起QBC939和RBE细胞中PARP和Caspase-3的活化剪切;而且在体内实验中,Western blot结果证实sal或rap单独干预未引起荷瘤的QBC939中PARP和Caspase-3的活化剪切,而联合应用时引起了PARP和Caspase-3明显的活化剪切,并且sal处理可以消除rap诱导的p-AKT和Bcl-x L表达上调;用Bcl-x L抑制剂ABT-737处理后抑制QBC939细胞成瘤,并且ABT-737和rap联合应用比ABT-737或rap单独干预表现出对QBC939细胞成瘤的更强抑制效应;Western blot结果表明在QBC939成瘤组织中,ABT-737和rap联合应用引起了PARP和Caspase-3明显的活化剪切;Western blot结果证实,ABT-737和rap联合应用在培养的QBC939细胞中引起了PARP和Caspase-3明显的活化剪切。结论:Sal和rap联合应用对胆管癌细胞有协同的抑制效应;Sal和rap可以部分通过调控p-Akt和Bcl-x L表达实现对胆管癌细胞的协同抑制效应。
[Abstract]:Aim: to study the inhibitory effect of eukaryotic translation initiation factors2e IF2 伪 inhibitor salubrinalen and rapamycin rapin on human cholangiocarcinoma cells and its molecular mechanism. Methods: human cholangiocarcinoma cell lines QBC939 and RBE were cultured. Rap was used to inhibit m TOR signaling pathway of cholangiocarcinoma cells. Sal, an e IF2 伪 inhibitor, was used to treat Bcl-x L inhibitor ABT-737 alone or with rap alone or in combination with rap. QBC939 was injected subcutaneously into nude mice to carry out tumor implantation, and then the tumor bearing effect was monitored by adding corresponding drugs to different experimental groups. The expression levels of related molecules and apoptosis-related molecules (PARP- Cleaved Caspase-3) in tumor cells and tumor-bearing tissues after drug intervention were analyzed by Western blotting. The proliferation rate of cells was measured by CCK-8 and the effect of drug intervention on the proliferation of mouse tumor bearing tissues was analyzed by immunohistochemistry. Results Rap inhibited the proliferation of CCA cells: Western blot analysis showed that rap could inhibit the activation of m TOR pathway in CCA cells. CCK-8 assay showed that rap inhibited the proliferation of QBC939 and RBE cells. Moreover, it did not induce the apoptosis of QBC939 and RBE cells in a time-dependent manner. (the results showed that the e IF2 伪 / ATF4 signaling pathway was overactivated in CCA cells. In addition, sal could inhibit the activity of e IF2 伪. CCK-8 assay confirmed that sal inhibited the proliferation of QBC939 and RBE cells. In addition, Sal did not induce the apoptosis of QBC939 and RBE cells. (data did not show that Sal inhibited the tumorigenesis of CCA cells: in vivo, it inhibited the tumor-bearing ability of QBC939 in nude mice. The inhibitory effect of sal on the proliferation of QBC939 cells in vivo was confirmed by immunohistochemistry. The combined use of rap and Sal inhibited the tumorigenesis of CCA cells. Sal and rap alone intervened to inhibit the proliferation of QBC939 cells in nude mice. The mechanism of synergistic inhibition of sal and rap on the tumorigenesis of QBC939 cells combined with rap: immunohistochemical results showed that the combined intervention of sal and rap showed stronger inhibitory effect on the growth of QBC939 cells bearing tumor than sal or rap alone. The results of CCK-8 analysis in vitro suggested that sal and rap had synergistic inhibitory effects on the proliferation of QBC939 cells. Western blot analysis showed that sal treatment could not only down-regulate the expression of p-AKT but also eliminate the up-regulation of p-AKT expression induced by rap in QBC939 cells. The results of Western blot showed that sal and rap alone or in combination did not induce the activation of PARP and Caspase-3 in QBC939 and RBE cells. Furthermore, the results of Western blot showed that sal or rap alone interfered with the activated shear of PARP and Caspase-3 in QBC939 without tumor, but the combination of PARP and Caspase-3 resulted in obvious activated shearing of PARP and Caspase-3. Sal treatment could eliminate the up-regulation of p-AKT and Bcl-x L expression induced by rap, and Bcl-x L inhibitor ABT-737 could inhibit the proliferation of QBC939 cells. The combination of ABT-737 and rap showed stronger inhibitory effect on QBC939 cell carcinogenesis than that of ABT-737 or rap alone. The results showed that the combination of ABT-737 and rap in QBC939 tumorigenic tissue resulted in obvious results of PARP and Caspase-3 activated shearing Western blot. It was confirmed that the combination of ABT-737 and rap induced the activation of PARP and Caspase-3 in cultured QBC939 cells. Conclusion there is a synergistic inhibitory effect of rap and Sal on cholangiocarcinoma cells. Sal and rap can partly regulate the expression of p-Akt and Bcl-x L to achieve the synergistic inhibitory effect on cholangiocarcinoma cells.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.8

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