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目的:观察小干扰RNA(siRNA)对结肠癌相关基因PTEN表达的抑制作用及其对结肠癌细胞增殖能力的影响。方法:构建携带有针对PTEN基因序列的siRNA真核绿色荧光表达载体重组质粒pGPU6/GFP/Neo-PTEN-1、pGPU6/GFP/Neo-PTEN-2、pGPU6/GFP/Neo-PTEN-3和pGPU6/GFP/Neo-PTEN-4,脂质体法转染入结肠癌HCT-8细胞(分别为p1、p2、p3和p4组),同时设转染阴性对照质粒组(p5组)和亲本细胞组(p6组),实时PCR方法检测PTEN mRNA表达,MTT法分析细胞增殖活性变化。结果:通过荧光显微镜观察计数,细胞转染率为58%。实时PCR方法检测结果显示,siRNA重组质粒pGPU6/GFP/Neo-PTEN-1、pGPU6/GFP/Neo-PTEN-2、pGPU6/GFP/Neo-PTEN-3和pGPU6/GFP/Neo-PTEN-4的抑制率分别为48.3%、76.5%、29.4%和61.2%,与p6组比较差异均有统计学意义(P<0.05)。MTT结果表明,细胞增殖抑制能力明显增强。结论:PTEN基因在肿瘤的发生发展中可能起到一定的作用。
Objective: To observe the inhibitory effect of small interfering RNA (siRNA) on the expression of PTEN in colon cancer and its effect on the proliferation of colon cancer cells. Methods: Recombinant plasmids pGPU6 / GFP / Neo-PTEN-1, pGPU6 / GFP / Neo-PTEN-2, pGPU6 / GFP / Neo-PTEN-3 and pGPU6 harboring siRNA targeting PTEN gene were constructed. / GFP / Neo-PTEN-4 was transfected into colon cancer HCT-8 cells (p1, p2, p3 and p4 respectively) by Lipofectamine 2000. The negative control plasmid group (P6 group). The expression of PTEN mRNA was detected by real-time PCR. The cell proliferation activity was analyzed by MTT assay. Results: The number of transfected cells was 58% by fluorescence microscopy. The results of real-time PCR assay showed that siRNA recombinant plasmids pGPU6 / GFP / Neo-PTEN-1, pGPU6 / GFP / Neo-PTEN-2, pGPU6 / GFP / Neo-PTEN-3 and pGPU6 / GFP / Neo-PTEN-4 The inhibitory rates were 48.3%, 76.5%, 29.4% and 61.2%, respectively, which were significantly different from those of p6 group (P <0.05). MTT results show that cell proliferation inhibitory capacity increased significantly. Conclusion: PTEN gene may play a role in the development of tumors.