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[目的]观察siRNA沉默Bmi-1表达对Hela细胞增殖能力的影响。[方法]构建了表达Bmi-1 siRNA的重组真核表达载体,将其转染入Hela细胞,利用荧光法观察转染效率。用RT-PCR及Western blot方法检测转染后细胞Bmi-1 mRNA及Bmi-1蛋白的表达情况;用MTT比色法、台盼蓝拒染法检测Bmi-1 siRNA对Hela细胞增殖的抑制作用;用流式细胞仪分析各组细胞的细胞周期;用平板克隆形成实验检测Bmi-1 siRNA对Hela细胞的单细胞增殖能力的影响;应用免疫细胞化学(SP法)及Western blot法检测各组细胞Ki-67及CyclinD1的表达。[结果]将构建好的重组质粒成功转染进入了Hela细胞中且转染效率在90%左右;Bmi-1 siRNA转染Hela细胞Bmi-1 mRNA及Bmi-1蛋白表达沉默,Bmi-1表达沉默后抑制Hela细胞增殖并使细胞周期阻滞于G0-G1期,能明显抑制Hela细胞的单细胞增殖能力;同时Ki-67及CyclinD1的表达均明显下降。[结论]siRNA介导的Bmi-1基因的表达沉默能抑制Hela细胞的增殖能力,Bmi-1表达可能与宫颈癌的发生发展相关。
[Objective] To observe the effect of siRNA silencing Bmi-1 expression on the proliferation of Hela cells. [Method] The recombinant eukaryotic expression vector expressing Bmi-1 siRNA was constructed and transfected into Hela cells. The transfection efficiency was observed by fluorescence method. The expression of Bmi-1 mRNA and Bmi-1 protein were detected by RT-PCR and Western blot. The inhibitory effect of Bmi-1 siRNA on Hela cell proliferation was detected by MTT assay and trypan blue exclusion assay The cell cycle of each group was analyzed by flow cytometry. The effect of Bmi-1 siRNA on single cell proliferation of Hela cells was detected by plate clone formation assay. Immunocytochemistry (SP) and Western blot were used to detect the expression of Bmi- Cell Ki-67 and CyclinD1 expression. [Result] The constructed recombinant plasmid was successfully transfected into Hela cells and the transfection efficiency was about 90%. The expression of Bmi-1 mRNA and Bmi-1 protein in Bmi-1 siRNA transfected Hela cells was silenced, and the expression of Bmi-1 After silencing, the proliferation of Hela cells was inhibited and the cell cycle was arrested in G0-G1 phase. The single cell proliferation ability of Hela cells was significantly inhibited. Meanwhile, the expressions of Ki-67 and CyclinD1 were significantly decreased. [Conclusion] The silencing of siRNA mediated Bmi-1 gene can inhibit the proliferation of Hela cells. The expression of Bmi-1 may be related to the occurrence and development of cervical cancer.