过表达高尔基体α-甘露糖苷酶Ⅱ对人胃癌BGC-823细胞凋亡的影响

Effect of over-expression of Golgi α-mannosidase Ⅱ on apoptosis of human gastric cancer BGC823 cells

目的探讨过表达高尔基体α-甘露糖苷酶Ⅱ(Golgiα-mannosidaseⅡ,GMⅡ)对人胃癌BGC-823细胞凋亡的影响。方法构建GMⅡ基因真核过表达质粒EX-E2372-M03,通过脂质体Lipofectamine 2000转染BGC-823细胞,用400 mg/LG418筛选稳定转染的细胞。采用RT-PCR和Western blot分别检测转染后细胞中GMⅡ基因mRNA的转录水平以及蛋白的表达水平,Hoechst 33258荧光染色法和流式细胞术(Annexin V/PI双染)检测过表达GMⅡ后细胞的凋亡情况。结果转染重组质粒EX-E2372-M03后,BGC-823细胞GMⅡ基因mRNA的转录水平和蛋白的表达水平明显增加(P<0.05);细胞凋亡率明显降低(P<0.05)。结论过表达GMⅡ可能通过抑制胃癌细胞的凋亡,进而促进胃癌的发生发展。

Objective To investigate the effect of over-expression of Golgi α-mannosidase Ⅱ（GMⅡ）on the apoptosis of human gastric cancer BGC-823 cells.Methods Eukaryotic over-expression vector EX-E2372-M03 for GMⅡ gene was constructed and transfected to BGC-823 cells in mediation of Lipofectamine 2000.The stably transfected BGC-823 cells were screened with 400 mg / L G418,and determined for transcription level of GM Ⅱ mRNA by RT-PCR,for expression level of GM Ⅱ protein by Western blot,and for apoptosis after over-expression of GMⅡ by Hoechst 33258 fluorescent staining and flow cytometry（Annexin V / PI staining）.Results Both the transcription level of GMⅡ mRNA and expression level of GMⅡ protein increased significantly in BGC-823 cells after transfection with EX-E2372-M03（P 〈 0.05）,while the apoptosis rate of cells decreased significantly（P 〈 0.05）.Conclusion The overexpression of GMⅡ gene might promoted the onset and progress of gastric cancer by inhibiting the apoptosis of gastric cancer cells.