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.docVIP

PAGE PAGE 3 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 杨紫汐，易永芬，杨雅莹（400016 重庆，重庆医科大学病理学教研室，分子医学与肿瘤研究中心） [摘要]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结果显示过表达GMⅡ后胃癌细胞增殖活性明显高于对照组(P＜0.05)。流式检测显示，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。 [关键词] 高尔基体α-甘露糖苷酶Ⅱ；C-myc；Bcl-2；胃癌；增殖 [中图法分类号] [文献标志码]A Effect of overexpressed GMⅡ on proliferation of gastric cell line BGC-823 Yang Zixi, Yi Yongfen, Yang Yaying (Department of Pathology, Molecular Medicine and Tumor Research Center, Chongqing Medical University, Chongqing 400016,China) [Abstract] Objective To construct the eukaryotic expression plasmid of human Golgi α-mannosidase Ⅱ(GMⅡ), identify its expression in gastric cancer cell line BGC-823, and investigate its effect on proliferation of the cells. Methods The eukaryotic overexpressed plasmid vector EX-E2372-M03 was constructed, then transfected to BGC-823 with lipofectamine, and the transfection efficiency was proved by Fluorescence Microscopy. RT-PCR were used to determine the mRNA expressions of GMⅡ、c-myc、bcl-2 after transfection. Flow cytometry and MTT were used to determine the effect of overexpressed GMⅡ on cell cycle distribution and proliferation activity. Western blot were used to detect the protein expressions of GMⅡ、c-myc and bcl-2 after transfection.Results The eukaryotic expression plasmid of GMⅡ was successfully constructed, which were validated by DNA sequence analysis. RT-PCR analysis indicated that mRNA expression of GMⅡ and c-myc was up-regulated significantly(P＜0.05) in gastric cancer cell line BGC-823 after transfection with EX-E2372-M03. Compare to the control g