论文部分内容阅读
目的:观察RNA干扰技术能否有效抑制非小细胞肺癌细胞株A549细胞中Polo-like激酶1(Plk1)的表达水平,以及抑制后对A549细胞生长的影响。方法:运用脂质体法,以Plk1为靶点,构建能产生siRNA的质粒载体psiRNA-hH1-Plk1并转入A549细胞。RT-PCR检测Plk1 mRNA表达的变化、Western blotting检测Plk1、cyc-linB1、p53蛋白的表达变化、细胞计数分析细胞增殖、流式细胞术分析细胞周期变化和凋亡、免疫荧光染色检测α微管蛋白的表达。结果:psiRNA-hH1-Plk1质粒能特异地抑制Plk1基因的表达并使其活性下降,致使cyclinB1及p53蛋白的表达水平升高,微管聚集障碍或形成单极的纺锤体;A549细胞增殖减慢,出现G2/M期阻滞和凋亡。结论:上述结果提示针对Plk1基因的RNA干扰有望用于肿瘤的基因治疗。
Objective: To observe whether RNAi can effectively inhibit the expression of Plk1 in non-small cell lung cancer cell line A549 and the effect of inhibiting it on the growth of A549 cells. Methods: Lipopolysaccharide was used as a target to construct siRNA vector plasmid psiRNA-hH1-Plk1 and transfected into A549 cells. The changes of Plk1 mRNA expression were detected by RT-PCR, the expression of Plk1, cyclinB1 and p53 protein were detected by Western blotting, the cell proliferation was analyzed by cell counting, the cell cycle was analyzed by flow cytometry and the apoptosis was detected by immunofluorescence staining Protein expression. Results: psiRNA-hH1-Plk1 plasmid could specifically inhibit the expression of Plk1 gene and decrease its activity, resulting in the increase of cyclinB1 and p53 protein expression, microtubule aggregation disorder or unipolar spindle formation; A549 cell proliferation slowed down , There G2 / M phase arrest and apoptosis. Conclusion: The above results suggest that the RNA interference targeting Plk1 gene is expected to be used in the gene therapy of tumors.