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目的探讨胃癌发展的分子机制,通过构建ERK1/2shRNA真核表达载体,体外研究其对人胃癌细胞株BGC823ERK1/2蛋白及DcR3表达水平的影响。方法应用PRNAT-U6.1/Neo载体构建ERK1/2基因shRNA重组质粒,经脂质体法导入BGC823细胞中,分别设置对照组、干扰组和U0126抑制剂组。Westernblot法检测转染后BGC823细胞及抑制剂使用后ERK1/2蛋白的表达变化,荧光显微镜检测质粒自带GFP基因的表达情况确认转染效率,ELISA法检测各组细胞上清中DcR3分泌蛋白的表达特点。结果成功构建ERK1/2基因shRNA重组质粒。证明了ERK1/2蛋白的表达与DcR3的分泌水平在BGC823细胞株中呈正相关。结论 ERK1/2干扰质粒明显降低BGC823细胞的ERK1/2蛋白表达水平,ERK信号通路对DcR3的分泌具有重要调控作用,为其下游调控机制的研究奠定了基础。
OBJECTIVE: To investigate the molecular mechanism of gastric cancer development and to investigate the effect of ERK1 / 2 shRNA on the expression of BGC823ERK1 / 2 and DcR3 in vitro. Methods shRNA recombinant plasmid of ERK1 / 2 gene was constructed by PRNAT-U6.1 / Neo vector and then transfected into BGC823 cells by liposome. The control group, interference group and U0126 inhibitor group were set up respectively. The expression of ERK1 / 2 protein in BGC823 cells and its inhibitor after transfection was detected by Western blot. The expression of GFP gene was detected by fluorescence microscopy to confirm the transfection efficiency. The secretion of DcR3 in the supernatant of each group was detected by ELISA Expression characteristics. Results The shRNA recombinant plasmid of ERK1 / 2 gene was successfully constructed. It was proved that the expression of ERK1 / 2 protein was positively correlated with the secretion of DcR3 in BGC823 cell line. Conclusion The ERK1 / 2 interference plasmid can significantly decrease the expression of ERK1 / 2 protein in BGC823 cells. ERK signaling pathway plays an important role in regulating the secretion of DcR3, which lays the foundation for its downstream regulatory mechanism.