过表达Mxi1对人胃癌SGC-7901细胞增殖和凋亡的影响  

Effects of overexpression of Mxi1 on cell proliferation and apoptosis of human gastric cancer SGC-7901 cells

作　　者：胡圳圳[1] 蒋秀琴[1] 许金金[1] 郭文文[1] 郑大同[1] 

机构地区：[1]南京医科大学第二附属医院临床分子基因检测中心,江苏南京210003

出　　处：《南京医科大学学报（自然科学版）》2017年第4期423-427,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81301822);南京医科大学科技发展基金(2012NJMU088)

摘　　要：目的:构建含有Max结合蛋白1(Mxi1)基因的重组慢病毒载体,并探讨过表达Mxi1基因对人胃癌SGC-7901细胞增殖和凋亡的影响。方法:利用DNA重组技术将Mxi1基因克隆至慢病毒载体,经酶切和测序鉴定后,将重组质粒与慢病毒辅助包装元件质粒共转染293T细胞,获得含Mxi1基因的重组慢病毒。重组慢病毒感染人胃癌SGC-7901细胞,荧光显微镜下观察感染效率,逆转录聚合酶链反应(RT-PCR)检测Mxi1、cyclinB1和caspase-8的基因表达,免疫印迹法(Western blot)检测Mxi1蛋白的表达。CCK-8比色法和流式细胞术分别检测Mxi1基因对人胃癌SGC-7901细胞增殖和凋亡的影响。结果:成功构建含Mxi1基因的慢病毒表达载体,RT-PCR和Western blot检测到Mxi1基因和蛋白的表达。RT-PCR结果显示,过表达Mxi1的SGC-7901细胞与空白对照细胞及过表达空病毒对照细胞相比,细胞内cyclinB1的mRNA表达水平明显降低,而caspase-8的mRNA表达水平显著升高。CCK-8实验和流式细胞术检测结果显示,过表达Mxi1的SGC-7901细胞与空白对照细胞及过表达空病毒对照细胞相比,细胞增殖活性明显降低,细胞凋亡率显著升高。结论:成功构建Mxi1慢病毒载体且有效转染SGC-7901细胞,过表达Mxi1可以抑制胃癌细胞增殖并促进其凋亡。Objective：To construct a lentiviral vector containing Mxil gene and observe the effect of Mxil on proliferation and apoptosis of SGC-7901 cells. Methods：The Mxil gene was cloned into lenfiviral expression vector by recombining DNA technology. The recombinant plasmid was identified by restriction enzyme digestion and gene sequencing, and then cotransfected 293T cells with the auxiliary packaging components plasmids to obtain recombinant lentivirus containing Mxil gene.SGC-7901 cells were infected with the recombinant lentivirus,and the infection efficiency was observed under fluorescence microscope.Expressions of Mxil ,cy- elinB1 and caspase-8 gene were identified by RT-PCR and expression of Mxil protein was identified by Western blot. The prolifera- tion and apoptosis of SGC-7901 cells were examined by CCK-8 and flow cytometry,respectively.Results：The Mxil gene was success- fully cloned to lentiviral,expressions of Mxil gene and protein were confirmed by RT-PCR and Western blot.Mxil-overexpressed SGC- 7901 cells expressed less cyclinB1 and more caspase-8 compared to blank control cells and empty vector-overexpressed cells con- finned by RT-PCR.CCK-8 and flow cytometry experimental results showed that the proliferation ability of Mxil-overexpressed SGC- 7901 cells was deteriorated significantly compared to blank control cells and empty vector-overexpressed cells,and the apoptosis rate of Mxil-overexpressed cells was higher than that of blank control ceils and empty vector-overexpressed cells.Conclusion：The Mxil recombinant lentiviral vector had been successfully constructed and effectively transfected SGC-7901 cells.Mxil could inhibit the pro- liferation and promote the apoptosis of SGC-7901 cells.

关 键 词：Max结合蛋白1 人胃癌SGC-7901细胞 增殖 凋亡 慢病毒载体 

分 类 号：R735.2[医药卫生—肿瘤]