本文選題：HSP90　切入點：Ganetespib　出處：《江蘇大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:第二代HSP90(heat shock protein 90)抑制劑(Ganetespib)靶向治療套細胞淋巴瘤(mantel cell lymphoma,MCL)的作用和機制研究,為Ganetespib用于治療MCL患者提供實驗依據(jù),也為HSP90受到小分子抑制劑抑制后,參與MCL發(fā)生相關的蛋白信號調控機制研究奠定基礎。方法:首先,我們研究了Ganetespib對常見的兩種MCL細胞株Jeko-1和Granta-519以及正常人外周血分離的淋巴細胞TZG-1的增殖影響。然后,再應用流式細胞儀,分析Jeko-1和Granta-519細胞經(jīng)過Ganetespib預處理后,細胞周期的變化情況。接著又分析了MCL細胞株Jeko-1經(jīng)Ganetespib預處理后,細胞凋亡的變化情況。此外,我們還運用了STRING軟件,分析與HSP90關系緊密的蛋白。將關聯(lián)系數(shù)在0.4以上的蛋白作為進一步研究對象,設計了編碼這些蛋白基因對應的mRNA引物,通過qRT-PCR檢測mRNA的水平。再用western blot檢測在Ganetespib預處理后,編碼該蛋白的基因對應的mRNA顯著下調的幾個蛋白(ERBB2、MYC、EGFR、AKT1和BCL2)。另外,我們還將Ganetespib作用于皮下接種了Jeko-1細胞的免疫缺陷的Nu/Nu小鼠上。取下的腫瘤組織切片后,采用免疫組織化學染色,來確定腫瘤組織細胞的凋亡情況和一些致癌蛋白含量的變化。最后,我們從醫(yī)院收集了幾種不同淋巴瘤患者病人的淋巴細胞,檢測了Ganetespib抑制這些細胞的增殖情況和兩種致瘤蛋白含量的變化。結果:體外實驗中,Ganetespib在有效抑制Jeko-1和Granta-519增殖的同時,對TUZ-1細胞的殺傷較小。Ganetespib能夠促進Jeko-1和Granta-519細胞G2/M期的停滯,抑制細胞增殖。Ganetespib能呈劑量依賴性地誘導Jeko-1細胞的凋亡。經(jīng)Ganetespib預處理的Jeko-1細胞,抑癌蛋白CDH1上調,致癌蛋白ERBB2、MYC、EGFR、AKT1和BCL2下調。小鼠體內實驗中,Ganetespib能夠有效抑制小鼠側翼移植的Jeko-1細胞的腫瘤生長,并且促進腫瘤組織細胞的凋亡。其中腫瘤組織內的兩種致癌蛋白(cyclin D1和c-Myc)含量也顯著下調。臨床上,Ganetespib也能有效抑制不同淋巴瘤患者的淋巴細胞增殖,c-Myc和c-Jun兩種致癌蛋白的含量也明顯下調。結論:以上結果表明,Ganetespib能夠有效抑制MCL細胞株的增殖,促進MCL細胞株的凋亡。HSP90功能受到抑制后,抑癌蛋白上調,致癌蛋白下調。Ganetespib也能有效抑制MCL細胞株皮下移植的小鼠腫瘤的生長,還能抑制臨床分離的淋巴瘤患者的淋巴細胞增殖。
[Abstract]:Objective: to investigate the effect and mechanism of the second generation HSP90(heat shock protein 90 inhibitor, Ganetespib, in the treatment of mantle cell lymphoma (MCLs), and to provide experimental evidence for the use of Ganetespib in the treatment of MCL patients and the inhibition of HSP90 by small molecular inhibitors. Methods: firstly, we studied the effects of Ganetespib on the proliferation of two common MCL cell lines, Jeko-1 and Granta-519, and the TZG-1 of lymphocytes isolated from normal human peripheral blood. Flow cytometry was used to analyze the changes of cell cycle of Jeko-1 and Granta-519 cells after pretreatment with Ganetespib. Then, the changes of apoptosis of MCL cell line Jeko-1 after pretreatment with Ganetespib were analyzed. In addition, we also used STRING software. The protein closely related to HSP90 was analyzed. The protein with correlation coefficient above 0.4 was used as the further research object. The corresponding mRNA primers encoding these protein genes were designed, and the level of mRNA was detected by qRT-PCR. Western blot was used to detect the level of mRNA after Ganetespib pretreatment. Several proteins, ERBB2My MYCN-EGFRN AKT1 and BCL2, were significantly down-regulated by mRNA corresponding to the gene encoding this protein. In addition, Ganetespib was subcutaneously inoculated into Nu/Nu mice with immunodeficient Jeko-1 cells. The tumor sections removed were stained by immunohistochemistry. To determine the apoptosis of tumor cells and the changes in the contents of some carcinogenic proteins. Finally, we collected lymphocytes from several different types of lymphoma patients. Results: in vitro, Ganetespib inhibited the proliferation of Jeko-1 and Granta-519 and inhibited the proliferation of TUZ-1 cells. Ganetespib could promote the arrest of G _ 2 / M phase in Jeko-1 and Granta-519 cells. Inhibiting cell proliferation. Ganetespib could induce apoptosis of Jeko-1 cells in a dose-dependent manner. Jeko-1 cells pretreated with Ganetespib up-regulated the tumor suppressor protein CDH1. The carcinogenic protein ERBB2, MYC, EGFRN, AKT1 and BCL2 are down-regulated. Ganetespib can effectively inhibit the growth of Jeko-1 cells in vivo. The levels of cyclin D1 and c-Myc) in tumor tissues were also significantly down-regulated. Ganetespib could also effectively inhibit the proliferation of lymphocytes in patients with different lymphomas induced by c-Myc and c-Jun. Conclusion: these results suggest that Ganetespib can effectively inhibit the proliferation of MCL cells. After the inhibition of MCL cell line apoptosis. HSP90 function was inhibited, tumor suppressor protein was up-regulated, and carcinogenic protein down-regulated. Ganetespib could also effectively inhibit the growth of mouse tumor transplanted subcutaneously by MCL cell line, and also inhibit the lymphocyte proliferation of clinically isolated lymphoma patients.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96

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