【摘要】 目的探讨P38丝裂原活化蛋白激酶(P38MAPK)的短发夹环RNA(shRNA)质粒对H9C-2心肌细胞增殖变化的影响并探讨其发生机制。方法构建3种P38MAPK shRNA质粒并测序鉴定,将其转染入H9C-2心肌细胞中,应用RT-PCR和Western blotting检测心脏细胞P38MAPK mRNA和蛋白的表达。结果P38MAPK shRNA质粒分布在心肌细胞的胞浆及细胞核中,与心肌细胞组比较,AngⅡ组和shRNA阴性组的P38MAPK mRNA和蛋白水平显著升高(P<0.01,P<0.01);与AngⅡ组比较,shRNA1、shRNA2和shRNA3组的P38MAPK mRNA和蛋白水平明显降低(P<0.05,P<0.05,P<0.01)。结论 P38MAPK shRNA质粒成功转染心肌细胞,P38MAPK shRNA3质粒能最有效地抑制心肌细胞P38MAPK的表达。更多还原

【Abstract】 【Objective】 To evaluate the effects of P38 mitogen-activated protein kinase(MAPK) short hair RNA(shRNA) plasmid delivery by Lipofectamin on mRNA and protein expression in H9C-2 myocardial cells.【Methods】 Three P38MAPK shRNA sequences were designed and synthesized,and subcloned into GPU6/ GFP/Neo plasmid.After restriction enzyme digestion and sequencing,the selected positive recombinant plasmid was transfected into H9C-2 myocardial cells.The mRNA and protein expressions of P38MAPK in H9C-2 myocardial cells were analyzed by RT-PCR and Western blotting analysis respectively.【Results】 It showed that the P38MAPK shRNA GPU6/GFP/Neo plasmid was taken up into H9C-2 myocardial cells.Compared with those in the H9C-2 myocardial cell group,the P38MAPK mRNA and protein levels in Ang Ⅱ group and plasmid control group significantly increased(P <0.01,P <0.01;respectively).Compared with those in the Ang Ⅱ group,the P38MAPK mRNA and protein levels in shRNA1,shRNA2 and shRNA3 group significantly decreased(P <0.05,P <0.05,P <0.01;respectively).【Conclusion】 P38MAPK shRNA plasmid could be transfected into cultured myocardial cells triumphally.P38MAPK shRNA3 is the most effective shRNA which inhibited the level of P38MAPK mRNA and protein expression.更多还原