核糖核酸干扰抑制心肌细胞KCNJ2基因表达

Inhibition of Gene KCNJ2 Expression by RNA Interference

目的:构建抑制大鼠心肌细胞KCNJ2基因表达的真核表达质粒,应用RNA干扰技术观察其抑制心肌细胞KCNJ2基因表达的情况,为生物起搏器的研究提供一种新的思路和方法。方法:选择5个针对大鼠心肌细胞基因KCNJ2mRNA的干扰位点,构建含5个目的基因的重组质粒pEGFP61kir2.1。实验组:乳鼠心肌细胞转染pEGFP61kir2.1质粒,阴性质粒对照组:转染PEGFP6IC,空白对照组:未做任何处理。转染大鼠心肌细胞后72h,通过逆转录多聚酶链反应(RTPCR)和Westernblot,在mRNA和蛋白质水平检测RNA干扰(RNAi)抑制KCNJ2基因表达的效果,并观察细胞搏动频率变化。结果:实验组心肌细胞KCNJ2mRNA和蛋白表达明显低于两对照组(P<0.01),两对照组无明显差异。转染后72h实验组搏动频率增加,显著高于两对照组(P<0.01),两对照组之间搏动频率无明显差异。结论:RNA干扰技术可用于抑制大鼠心肌细胞KCNJ2基因的表达,为生物起搏器的研究提供了一种新的思路和方法。

Objective：To develop a new method for the biological pacemaker study,anti to construct plasmid to suppress the expression of KCNJ2 gene in rat myocardial cells. Methods：Five RNAi sites targeting the rat gene KCNJ2 gene were selected, and 5 pairs of oligonuleotides fragments were designed and synthesized. The resuhing plasmid pEGFP6-1kir2. 1. was made to transfect the rat myocardial cells. After 72 hours RTPCR and Western-blot were used to detect the expression of the mRNA and protein. The beats of rat myocardial cells were recorded. Results：The expression of KCNJ2 gene was surpressed by the effective RNAi target. The beats of rat myocardial ceils were increased. Conclusions： RNAi can be used to suppress the expression of the KCNJ2 gene in rat myocardial cells. This experimant provides a new methos for the biological pacemaker study.