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CENP-I表达下调对HEK293细胞生长及染色体分离的影响

, PP. 1569-1572

Keywords: CENP-I基因,RNA干扰,细胞分裂指数

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Abstract:

目的构建CENP-I特异性RNA干扰真核表达载体,体外观察抑制CENP-I基因表达对HEK293细胞生长、细胞周期和染色体数目异常率的影响。方法构建CENP-IsiRNA真核表达载体,脂质体法将pGenesil-1空载体和pGenesil-1/CENP-I-siRNA-1、pGenesil-1/CENP-I-siRNA-2、pGenesil-1/CENP-I-siRNA-3真核表达载体分别导入人胚肾HEK293细胞。转染后24、48、72h收集细胞用Westernblot和FQ-PCR检测HEK293细胞内CENP-I蛋白及mRNA水平的表达情况,以确定干扰效果及最佳干扰时间。MTT法检测各实验组细胞生长情况,流式细胞术检测各组细胞的细胞周期分布,Giemsa染色法计数细胞分裂指数,常规法制备染色体标本。结果成功构建CENP-IsiRNA真核表达载体,筛选出具有干扰效果的质粒载体即pGenesil-1/CENP-I-siRNA-3,转染人胚肾HEK293细胞72h后可使CENP-I蛋白及mRNA表达显著下凋。CENP-I表达降低的HEK293细胞生长速度明显减慢(P<0.05),G2/M期细胞增加,分裂期细胞比例增大。结论RNA干扰有效地特异性抑制着丝粒特异性蛋白质CENP-I的表达。CENP-I的抑制可使人胚肾HEK293细胞生长减慢,增殖能力减弱,使细胞分裂期延长。

References

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