过表达高尔基体α-甘露糖苷酶Ⅱ对胃癌细胞株BGC-823增殖的影响

目的探讨过表达高尔基体α-甘露糖苷酶Ⅱ(Golgiα-mannosidaseⅡ,GMⅡ)对人胃癌细胞株BGC-823增殖活性的影响。方法构建真核表达载体EX-E2372-M03,通过脂质体Lipofectamine2000转染至人胃癌细胞株BGC-823,筛选稳定细胞株,用免疫荧光观察转染效率。用RT-PCR法检测转染细胞GMⅡ、c-myc、bcl-2 mRNA的表达,用Western blot法检测转染细胞GMⅡ、c-myc、bcl-2蛋白表达的变化,用MTT法及流式细胞仪检测过表达GMⅡ后对细胞增殖活性的影响。结果 GMⅡ真核表达质粒构建成功并成功转染,MTT结果示过表达GMⅡ后胃癌细胞增殖活性明显高于对照组(P<0.05)。流式细胞仪检测结果显示,与对照组相比较,GMⅡ过表达组胃癌细胞G1期细胞明显减少,S期细胞比例明显增加。转染后胃癌细胞GMⅡmRNA和蛋白表达水平明显高于对照组(P<0.05),且可明显上调胃癌细胞BGC-823中c-myc的mRNA和蛋白表达水平(P<0.05),但对bcl-2的调节作用不明显。结论过表达GMⅡ能促进胃癌细胞增殖,可能与上调c-myc的表达有关。

Effect of overexpressed Golgi α-mannosidaseⅡ on proliferation in human gastric cancer cell line BGC-823

Objective To investigate the effect of overexpressed Golgi α-mannosidaseⅡ(GMⅡ) on the proliferation in human gastric cancer cell line BGC-823.Methods Eukaryotic GMⅡ overexpression plasmid EX-E2372-M03 was constructed and transferred into human gastric cancer cell line BGC-823 with Lipofectamine 2000.The cell strains with stable expression were screened,and the transfection efficiency was determined by fluorescent microscopy.RT-PCR were used to determine the mRNA expressions of GMⅡ,c-myc and bcl-2 in transfected cells.Flow cytometry and MTT assay were used to determine the effects of overexpressed GMⅡ on cell cycle distribution and cell proliferation.Western blot analysis were used to detect the protein expression levels of GMⅡ,c-myc and bcl-2 in transfected cells.Results The mRNA and protein expression levels of GMⅡ increased significantly in gastric cancer cell line BGC-823 transfected with EX-E2372-M03(P<0.05),and the mRNA and protein expression levels of c-myc were upregulated by overexpressed GMⅡ,but those of bcl-2 were not significantly affected.The MTT results showed that the proliferation activity of the cells transfected with EX-E2372-M03 was significantly higher than that of the control cells(P<0.05).The flow cytometry results showed that compared with the control group,overexpressed GMⅡ led to a decrease in the percentage of cells in G1 phase(P<0.05),and an increase in that in S phase(P<0.05).Conclusion Overexpressed GMⅡ can promote the proliferation in gastric cancer cell line BGC-823,which may involve c-myc expression upregulation.