本文選題：HSP90　切入點(diǎn)：Ganetespib　出處：《江蘇大學(xué)》2017年碩士論文　論文類型：學(xué)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(xì)胞淋巴瘤(mantel cell lymphoma,MCL)的作用和機(jī)制研究,為Ganetespib用于治療MCL患者提供實(shí)驗(yàn)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(guān)的蛋白信號(hào)調(diào)控機(jī)制研究奠定基礎(chǔ)。方法:首先,我們研究了Ganetespib對(duì)常見(jiàn)的兩種MCL細(xì)胞株Jeko-1和Granta-519以及正常人外周血分離的淋巴細(xì)胞TZG-1的增殖影響。然后,再應(yīng)用流式細(xì)胞儀,分析Jeko-1和Granta-519細(xì)胞經(jīng)過(guò)Ganetespib預(yù)處理后,細(xì)胞周期的變化情況。接著又分析了MCL細(xì)胞株Jeko-1經(jīng)Ganetespib預(yù)處理后,細(xì)胞凋亡的變化情況。此外,我們還運(yùn)用了STRING軟件,分析與HSP90關(guān)系緊密的蛋白。將關(guān)聯(lián)系數(shù)在0.4以上的蛋白作為進(jìn)一步研究對(duì)象,設(shè)計(jì)了編碼這些蛋白基因?qū)?yīng)的mRNA引物,通過(guò)qRT-PCR檢測(cè)mRNA的水平。再用western blot檢測(cè)在Ganetespib預(yù)處理后,編碼該蛋白的基因?qū)?yīng)的mRNA顯著下調(diào)的幾個(gè)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(xì)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(xué)染色,來(lái)確定腫瘤組織細(xì)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(xì)胞,檢測(cè)了Ganetespib抑制這些細(xì)胞的增殖情況和兩種致瘤蛋白含量的變化。結(jié)果:體外實(shí)驗(yàn)中,Ganetespib在有效抑制Jeko-1和Granta-519增殖的同時(shí),對(duì)TUZ-1細(xì)胞的殺傷較小。Ganetespib能夠促進(jìn)Jeko-1和Granta-519細(xì)胞G2/M期的停滯,抑制細(xì)胞增殖。Ganetespib能呈劑量依賴性地誘導(dǎo)Jeko-1細(xì)胞的凋亡。經(jīng)Ganetespib預(yù)處理的Jeko-1細(xì)胞,抑癌蛋白CDH1上調(diào),致癌蛋白ERBB2、MYC、EGFR、AKT1和BCL2下調(diào)。小鼠體內(nèi)實(shí)驗(yàn)中,Ganetespib能夠有效抑制小鼠側(cè)翼移植的Jeko-1細(xì)胞的腫瘤生長(zhǎng),并且促進(jìn)腫瘤組織細(xì)胞的凋亡。其中腫瘤組織內(nèi)的兩種致癌蛋白(cyclin D1和c-Myc)含量也顯著下調(diào)。臨床上,Ganetespib也能有效抑制不同淋巴瘤患者的淋巴細(xì)胞增殖,c-Myc和c-Jun兩種致癌蛋白的含量也明顯下調(diào)。結(jié)論:以上結(jié)果表明,Ganetespib能夠有效抑制MCL細(xì)胞株的增殖,促進(jìn)MCL細(xì)胞株的凋亡。HSP90功能受到抑制后,抑癌蛋白上調(diào),致癌蛋白下調(diào)。Ganetespib也能有效抑制MCL細(xì)胞株皮下移植的小鼠腫瘤的生長(zhǎng),還能抑制臨床分離的淋巴瘤患者的淋巴細(xì)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(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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