本文選題：Salubrinal + 協(xié)同�。� 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:研究真核翻譯起始因子2α(eukaryotic translation initiation factors2,e IF2α)抑制劑salubrinal(sal)協(xié)同雷帕霉素(rapamycin,rap)對(duì)人膽管癌細(xì)胞的抑制效果及其分子機(jī)制。方法:培養(yǎng)人膽管癌細(xì)胞系QBC939和RBE細(xì)胞,用rap抑制膽管癌細(xì)胞m TOR信號(hào)通路,采用e IF2α抑制劑sal單獨(dú)或與rap聯(lián)合處理細(xì)胞、Bcl-x L抑制劑ABT-737單獨(dú)或與rap聯(lián)合處理細(xì)胞,通過向裸鼠皮下注射QBC939實(shí)現(xiàn)荷瘤,荷瘤后給不同實(shí)驗(yàn)組加相應(yīng)的藥物進(jìn)行干預(yù),監(jiān)測(cè)小鼠荷瘤效果,用免疫印跡分析藥物干預(yù)后腫瘤細(xì)胞和成瘤癌組織中相關(guān)分子以及凋亡相關(guān)分子PARP、Cleaved Caspase-3表達(dá)水平,用CCK-8檢測(cè)細(xì)胞增殖速率,免疫組化分析藥物干預(yù)對(duì)小鼠荷瘤組織增殖的影響。結(jié)果:(1)Rap抑制CCA細(xì)胞增殖:免疫印跡(Western blot)分析結(jié)果說明rap可以抑制CCA細(xì)胞中m TOR通路的活化,CCK-8檢測(cè)證實(shí)rap對(duì)QBC939和RBE細(xì)胞的增殖起抑制作用,并且呈時(shí)間依賴性抑制(P0.05);此外,rap未引起QBC939和RBE細(xì)胞明顯凋亡(數(shù)據(jù)未展示);(2)Sal抑制CCA細(xì)胞增殖:Western blot結(jié)果表明e IF2α/ATF4信號(hào)通路在CCA細(xì)胞中呈過度活化狀態(tài),并且sal可以抑制e IF2α的活性,CCK-8檢測(cè)證實(shí)sal對(duì)QBC939和RBE細(xì)胞的增殖起抑制作用,并且呈時(shí)間依賴性抑制(P0.05);此外,sal未引起QBC939和RBE細(xì)胞明顯凋亡(數(shù)據(jù)未展示);(3)Sal抑制CCA細(xì)胞成瘤:在體內(nèi)實(shí)驗(yàn)中,sal抑制QBC939在裸鼠體內(nèi)的荷瘤能力,并通過免疫組化證實(shí)sal抑制QBC939細(xì)胞體內(nèi)細(xì)胞增殖能力;(4)Sal和rap聯(lián)合應(yīng)用抑制CCA細(xì)胞成瘤:在裸鼠體內(nèi)sal和rap單獨(dú)干預(yù)抑制QBC939細(xì)胞的成瘤,二者聯(lián)合干預(yù)協(xié)同抑制QBC939細(xì)胞的成瘤;(5)Sal和rap聯(lián)合應(yīng)用協(xié)同抑制CCA細(xì)胞成瘤的機(jī)制:免疫組化證實(shí)sal和rap聯(lián)合干預(yù)比單獨(dú)sal或rap干預(yù)表現(xiàn)出對(duì)荷瘤的QBC939細(xì)胞生長更強(qiáng)的抑制作用;在體外實(shí)驗(yàn)中,CCK-8分析結(jié)果提示sal和rap對(duì)QBC939細(xì)胞增殖有協(xié)同抑制作用(P0.05)。用Western blot分析表明在QBC939細(xì)胞中sal處理不僅可以下調(diào)p-AKT的表達(dá)還可消除rap誘導(dǎo)的p-AKT表達(dá)上調(diào);在體外實(shí)驗(yàn)中,Western blot結(jié)果表明sal和rap單獨(dú)或聯(lián)合應(yīng)用未引起QBC939和RBE細(xì)胞中PARP和Caspase-3的活化剪切;而且在體內(nèi)實(shí)驗(yàn)中,Western blot結(jié)果證實(shí)sal或rap單獨(dú)干預(yù)未引起荷瘤的QBC939中PARP和Caspase-3的活化剪切,而聯(lián)合應(yīng)用時(shí)引起了PARP和Caspase-3明顯的活化剪切,并且sal處理可以消除rap誘導(dǎo)的p-AKT和Bcl-x L表達(dá)上調(diào);用Bcl-x L抑制劑ABT-737處理后抑制QBC939細(xì)胞成瘤,并且ABT-737和rap聯(lián)合應(yīng)用比ABT-737或rap單獨(dú)干預(yù)表現(xiàn)出對(duì)QBC939細(xì)胞成瘤的更強(qiáng)抑制效應(yīng);Western blot結(jié)果表明在QBC939成瘤組織中,ABT-737和rap聯(lián)合應(yīng)用引起了PARP和Caspase-3明顯的活化剪切;Western blot結(jié)果證實(shí),ABT-737和rap聯(lián)合應(yīng)用在培養(yǎng)的QBC939細(xì)胞中引起了PARP和Caspase-3明顯的活化剪切。結(jié)論:Sal和rap聯(lián)合應(yīng)用對(duì)膽管癌細(xì)胞有協(xié)同的抑制效應(yīng);Sal和rap可以部分通過調(diào)控p-Akt和Bcl-x L表達(dá)實(shí)現(xiàn)對(duì)膽管癌細(xì)胞的協(xié)同抑制效應(yīng)。
[Abstract]:Aim: to study the inhibitory effect of eukaryotic translation initiation factors2e IF2 偽 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 偽 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 偽 / ATF4 signaling pathway was overactivated in CCA cells. In addition, sal could inhibit the activity of e IF2 偽. 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.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.8

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