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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 recombinant lentiviral vector containing Max-binding protein 1 (Mxi1) gene and investigate the effect of overexpression of Mxi1 gene on the proliferation and apoptosis of human gastric cancer cell line SGC-7901. Methods: The Mxi1 gene was cloned into lentiviral vector by DNA recombination technology. After digestion and sequencing, the recombinant plasmids were co-transfected into 293T cells with lentivirus helper plasmids to obtain the recombinant lentivirus containing Mxi1 gene. The expression of Mxi1, cyclinB1 and caspase-8 were detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of Mxi1, cyclinB1 and caspase-8 were detected by Western blot. Protein expression. The effects of Mxi1 gene on proliferation and apoptosis of human gastric cancer cell line SGC-7901 were detected by CCK-8 assay and flow cytometry respectively. Results: The lentiviral vector containing Mxi1 gene was successfully constructed. The expression of Mxi1 gene and protein was detected by RT-PCR and Western blot. The results of RT-PCR showed that the expression of cyclinB1 mRNA and the expression of caspase-8 mRNA in SGC-7901 cells overexpressing Mxi1 were significantly lower than those in blank control cells and empty vector control cells. The results of CCK-8 assay and flow cytometry showed that the proliferation of SGC-7901 cells overexpressing Mxi1 was significantly lower than that of blank control cells and empty vector control cells, and the apoptosis rate was significantly increased. Conclusion: The Mxi1 lentiviral vector was successfully constructed and transfected into SGC-7901 cells successfully. Overexpression of Mxi1 can inhibit the proliferation and promote the apoptosis of gastric cancer cells.