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目的:研究RNA干扰(RNAi)对结肠癌HCT-8细胞IDH2基因表达的抑制作用及干扰后对HCT-8细胞增殖的影响,为结肠癌的基因靶向治疗提供理论依据。方法:实验分siRNA干扰质粒转染组、阴性质粒对照组和HCT-8细胞空白对照组。构建靶向基因IDH2的siRNA真核绿色荧光载体重组质粒PGPU6/GFP/Neo-IDH2,利用FuGENE HD转染剂转染HCT-8细胞,通过荧光定量PCR检测IDH2mRNA的表达情况及MTT法检测结肠癌HCT-8细胞增殖变化。结果:在荧光显微镜下进行细胞计数,细胞转染率为74%。实时荧光定量PCR检测,siRNA重组质粒PGPU6/GFP/Neo-IDH2对IDH2基因表达的抑制率为85.02%,与空白对照组比较差异有统计学意义(P<0.05)。MTT检测,干扰质粒转染组细胞的增殖能力明显低于空白对照组(P<0.05)。结论:抑制IDH2基因的表达可影响结肠癌细胞的增殖能力,提示IDH2基因在结肠癌的发生发展中起重要作用,抑制IDH2基因的表达可能成为一种治疗结肠癌的方法。
Objective: To study the inhibitory effect of RNA interference (RNAi) on the expression of IDH2 gene in human colon cancer HCT-8 cells and the effect of RNA interference on the proliferation of HCT-8 cells. Methods: The experiment was divided into siRNA interference plasmid transfection group, negative plasmid control group and HCT-8 cell blank control group. To construct the eukaryotic expression vector pGPU6 / GFP / Neo-IDH2 targeting the gene IDH2, transfected HCT-8 cells with FuGENE HD transfection reagent, detect the expression of IDH2 mRNA by fluorescence quantitative PCR and detect the colon cancer by MTT assay HCT-8 cell proliferation changes. Results: Cell counting was performed under a fluorescence microscope with a cell transfection rate of 74%. The real-time fluorescent quantitative PCR showed that the inhibitory rate of siRNA-recombinant plasmid PGPU6 / GFP / Neo-IDH2 on IDH2 gene expression was 85.02%, which was significantly lower than that of the blank control group (P <0.05). MTT assay showed that the proliferation ability of the transfected cells was significantly lower than that of the blank control group (P <0.05). Conclusion: Inhibition of IDH2 gene expression can affect the proliferation of colon cancer cells, suggesting that IDH2 gene plays an important role in the development of colon cancer. Suppression of IDH2 gene expression may become a method of treating colon cancer.