第二代HSP90抑制剂（Ganetespib）靶向治疗套细胞淋巴癌的作用和机制研究

发布时间：2018-03-13 13:18

本文选题：HSP90　切入点：Ganetespib　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:第二代HSP90(heat shock protein 90)抑制剂(Ganetespib)靶向治疗套细胞淋巴瘤(mantel cell lymphoma,MCL)的作用和机制研究,为Ganetespib用于治疗MCL患者提供实验依据,也为HSP90受到小分子抑制剂抑制后,参与MCL发生相关的蛋白信号调控机制研究奠定基础。方法:首先,我们研究了Ganetespib对常见的两种MCL细胞株Jeko-1和Granta-519以及正常人外周血分离的淋巴细胞TZG-1的增殖影响。然后,再应用流式细胞仪,分析Jeko-1和Granta-519细胞经过Ganetespib预处理后,细胞周期的变化情况。接着又分析了MCL细胞株Jeko-1经Ganetespib预处理后,细胞凋亡的变化情况。此外,我们还运用了STRING软件,分析与HSP90关系紧密的蛋白。将关联系数在0.4以上的蛋白作为进一步研究对象,设计了编码这些蛋白基因对应的mRNA引物,通过qRT-PCR检测mRNA的水平。再用western blot检测在Ganetespib预处理后,编码该蛋白的基因对应的mRNA显著下调的几个蛋白(ERBB2、MYC、EGFR、AKT1和BCL2)。另外,我们还将Ganetespib作用于皮下接种了Jeko-1细胞的免疫缺陷的Nu/Nu小鼠上。取下的肿瘤组织切片后,采用免疫组织化学染色,来确定肿瘤组织细胞的凋亡情况和一些致癌蛋白含量的变化。最后,我们从医院收集了几种不同淋巴瘤患者病人的淋巴细胞,检测了Ganetespib抑制这些细胞的增殖情况和两种致瘤蛋白含量的变化。结果:体外实验中,Ganetespib在有效抑制Jeko-1和Granta-519增殖的同时,对TUZ-1细胞的杀伤较小。Ganetespib能够促进Jeko-1和Granta-519细胞G2/M期的停滞,抑制细胞增殖。Ganetespib能呈剂量依赖性地诱导Jeko-1细胞的凋亡。经Ganetespib预处理的Jeko-1细胞,抑癌蛋白CDH1上调,致癌蛋白ERBB2、MYC、EGFR、AKT1和BCL2下调。小鼠体内实验中,Ganetespib能够有效抑制小鼠侧翼移植的Jeko-1细胞的肿瘤生长,并且促进肿瘤组织细胞的凋亡。其中肿瘤组织内的两种致癌蛋白(cyclin D1和c-Myc)含量也显著下调。临床上,Ganetespib也能有效抑制不同淋巴瘤患者的淋巴细胞增殖,c-Myc和c-Jun两种致癌蛋白的含量也明显下调。结论:以上结果表明,Ganetespib能够有效抑制MCL细胞株的增殖,促进MCL细胞株的凋亡。HSP90功能受到抑制后,抑癌蛋白上调,致癌蛋白下调。Ganetespib也能有效抑制MCL细胞株皮下移植的小鼠肿瘤的生长,还能抑制临床分离的淋巴瘤患者的淋巴细胞增殖。
[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.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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