靶向CDK2、cyclinE的siRNA对HepG_2细胞周期及凋亡的影响

EFFECTS OF siRNA TARGED CDK2 AND cyclinE ON CELL CYCLE AND APOPTOSIS OF HepG_2 CELLS

目的:探讨细胞周期蛋白依赖性激酶(cyclin-dependent-kinase 2,CDK2)活性对肝癌细胞株HepG2细胞周期和细胞凋亡的影响。方法:根据基因库中登录的人和鼠CDK2、cyclinE序列,设计并构建CDK2、cyclinE干扰RNA真核表达载体;脂质体法转染肝癌细胞株HepG2细胞,流式细胞术分析CDK2及cyclinE对HepG2细胞增殖的影响;蛋白质印迹法检测CDK2、cyclinE活性的变化caspase-3活性的影响。结果:1.成功构建CDK2及cyclinE干扰RNA真核表达载体psiCDK2、psiCyclinE,用脂质体法导入肝癌细胞株HepG2细胞中,有效表达。2.转染48h后与空载体组相比:psiCDK2、psiCyclinE组G1期细胞增多,G2/M和S期细胞减少;蛋白质印迹法分析表明psiCDK2、psiCyclinE组caspase-3酶原被激活。结论:靶向CDK2、cyclinE的siRNA能抑制HepG2细胞的增殖;靶向CDK2、cyclinE的siRNA能激活caspase-3,诱导肝癌细胞HepG2凋亡。

Objective：To observe the effect of CDK2 on the cell cycle and apoptosis of HepG2 cells. Methods：Designing the siCDK2 DNA template of CDK2 and cyclinE cloned into siRNA expression vector, transfecting the HepG2 cells by liposome, analysing the cell cycle of the transfected cells by flow cytometry,detecting the activation of caspase -3 by Western blotting in 48h. Results： Firstly,the siRNA expression vector psiCDK2 and psiCyclinE were constructed successfully and expressed effectively in transfeeted HepG2 cells. Secondly, the cells arrested in Gl stage were increased. The cells in G2/M and S stages were decreased after transfection in psiCDK2 and psiCyclinE transfected HepG2 cells compared with the control group. The caspase -3 was activated in psiCDK2 and psiCyclinE in transfected cells. Conclusion：The siRNA expression vector psiCDK2 and psiCyclinE were constructed successfully. The siRNA targeted CDK2 and cyclinE can inhibit the proliferation of HepG2. The siRNA of CDK2 and cyelinE can activate the easpase -3 and induce apoptosis of HepG2 cells.