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目的构建CD147干扰质粒,对肝癌细胞7721内源性CD147表达的抑制效果进行检测。方法设计及构建3对pGenesil-1-CD147/shRNA及1对阴性对照干扰质粒,通过测序鉴定。将干扰质粒用Lipofec-tamineTM2000转染肝癌细胞,通过瞬时转染获得细胞系,实时PCR和蛋白印迹法检测3对干扰质粒、阴性对照质粒的mRNA及蛋白表达水平。结果实时PCR和蛋白印迹法结果显示干扰质粒pGenesil-1-CD147/shRNA3对肝癌细胞7721内CD147mRNA及蛋白的抑制效果最显著。结论成功构建了CD147干扰真核表达载体,筛选出有效干扰质粒,为进一步研究低表达CD147的肝癌细胞7721在肝癌发生发展中的机制提供基础。
Objective To construct a CD147 interference plasmid to detect the inhibitory effect of endogenous CD147 expression in hepatocellular carcinoma cell line 7721. Methods Three pairs of pGenesil-1-CD147 / shRNA and one pair of negative control plasmids were designed and constructed and identified by sequencing. The interference plasmids were transfected into hepatoma cells with Lipofec-tamineTM2000, and the cell lines were obtained by transient transfection. The mRNA and protein expression levels of three pairs of interfering plasmids and negative control plasmids were detected by real-time PCR and Western blotting. Results Real-time PCR and Western blotting showed that the inhibitory effect of pGenesil-1-CD147 / shRNA3 on CD147 mRNA and protein in hepatocellular carcinoma cells 7721 was the most significant. Conclusion The eukaryotic expression vector of CD147 was successfully constructed and the plasmids were efficiently screened. This study may provide a basis for further study on the mechanism of hepatocellular carcinoma cell line 7721 with low expression of CD147.