通过反转录病毒侵染建立稳定过表达c-Myc的MIN6细胞系

发布时间：2018-03-01 21:08

本文关键词： c-Myc 反转录病毒载体过表达增殖凋亡　出处：《东北林业大学》2011年硕士论文　论文类型：学位论文

【摘要】：c-Myc为原癌基因,为正常组织器官发育过程中所必需。它主要通过特异或非特异性与靶基因DNA结合位点相结合,启动或抑制靶基因的表达,调节细胞生长、分化等生命活动。有研究发现,c-Myc基因在染色体上发生扩增或激活突变会引起细胞增殖加快,细胞侵袭能力加强,导致胰腺肿瘤的发生、发展及恶化；在p细胞中过表达c-Myc也可诱导细胞凋亡,减少β细胞群体数量。为了进一步研究c-Myc对于p细胞的调控机制,本研究通过用293GP细胞包装带有c-Myc基因的逆转录病毒载体,制备假性逆转录病毒,用其侵染胰腺β细胞系MIN6。通过对整合c-Myc基因的MIN6细胞G418筛选、扩增获得稳定过表达c-Myc基因的MIN6细胞克隆,并进一步分析c-Myc过表达引起MIN6细胞的生命活动变化。结果表明,通过抗性药物G418的筛选,获得整合逆转录病毒基因的多个MIN6细胞克隆RT-PCR检测结果表明,这些细胞能稳定过表达c-Myc基因,同时发现,在过表达c-Myc的M1N6细胞中可以检测到mtR-17-92基因簇的表达上调；通过分析过表达c-Myc克隆细胞的生长曲线,我们发现,过表达c-Myc会引起MIN6细胞生长加快,细胞周期变短；通过细胞染色,我们发现某些过表达c-Myc的MIN6细胞凋亡有明显增加。这些研究结果表明,在小鼠β细胞系中稳定过表达c-Myc会诱导MIN6细胞增殖加快、凋亡加剧,其机制有待于进一步的实验探索。
[Abstract]:C-Myc is a proto-oncogene that is necessary for the development of normal tissues and organs. It activates or inhibits the expression of target genes and regulates cell growth through the binding of specific or non-specific DNA binding sites to target genes. Some studies have found that the amplification or activation of c-Myc gene on chromosomes will accelerate cell proliferation, enhance the ability of cell invasion, and lead to the occurrence, development and deterioration of pancreatic tumors. Overexpression of c-Myc in p cells also induced apoptosis and reduced the number of 尾 cells. In order to further study the regulation mechanism of c-Myc on p cells, the retrovirus vector with c-Myc gene was packaged with 293GP cells. Pseudoretrovirus was prepared and infected with human pancreatic 尾 cell line MIN6.The MIN6 cell clones with stable overexpression of c-Myc gene were amplified by G418 screening of MIN6 cells integrated with c-Myc gene. Furthermore, the changes of life activity of MIN6 cells induced by c-Myc overexpression were further analyzed. The results showed that through the screening of the resistant drug G418, RT-PCR clones of multiple MIN6 cells integrating retrovirus gene were obtained. The results showed that these cells were able to overexpression c-Myc gene stably, and found that, Overexpression of c-Myc in M1N6 cells can be detected by up-regulation of mtR-17-92 gene cluster. By analyzing the growth curve of overexpression c-Myc clone cells, we found that overexpression of c-Myc can accelerate the growth of MIN6 cells and shorten cell cycle. We found that the apoptosis of some MIN6 cells with overexpression of c-Myc was significantly increased. These results indicated that overexpression of c-Myc in mouse 尾 cell lines could induce the proliferation and apoptosis of MIN6 cells, and the mechanism needed to be further explored.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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