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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。
Objective To investigate the effect of overexpression Golgiα-mannosidase Ⅱ (GMⅡ) on the proliferation of human gastric cancer cell line BGC-823. Methods The eukaryotic expression vector EX-E2372-M03 was constructed and transfected into human gastric cancer cell line BGC-823 by Lipofectamine 2000. Stable cell lines were screened and the transfection efficiency was observed by immunofluorescence. The expression of GMⅡ, c-myc and bcl-2 mRNA were detected by RT-PCR. The expressions of GMⅡ, c-myc and bcl-2 protein were detected by Western blot. MTT assay and flow cytometry The effect of over-expressing GMⅡ on cytotoxicity was detected by cytometry. Results The recombinant eukaryotic expression plasmid GMⅡ was successfully constructed and transfected successfully. MTT results showed that the proliferation activity of gastric cancer cells was significantly higher than that of the control group after overexpression of GMⅡ (P <0.05). Flow cytometry results showed that, compared with the control group, GMⅡ overexpression of gastric cancer cells in G1 phase cells decreased significantly, S phase cells increased significantly. The expression of GMⅡ mRNA and protein in gastric cancer cells was significantly higher than that in control group (P <0.05), and significantly up-regulated the mRNA and protein expression of c-myc in BGC-823 gastric cancer cells (P <0.05) 2 regulation is not obvious. Conclusion Overexpression of GMⅡ can promote the proliferation of gastric cancer cells, which may be related to the up-regulation of c-myc expression.