RNA干扰下调可溶性环氧化物水解酶表达对异丙肾上腺素诱导心肌细胞凋亡的影响

Inhibition of soluble epoxide hydrolase with RNA interference influence isoproterenol induced cardiomyocyte apoptosis

目的:构建含有可溶性环氧化物水解酶(sEH)基因特异性的RNA干扰序列质粒,选择性下调小鼠心肌细胞sEH的表达,筛选出抑制sEH效果最明显的表达质粒;观察其对异丙基肾上腺素(ISO)诱导的心肌细胞的凋亡及相关基因的影响。方法:构建2条靶向sEH基因的特异性小干扰RNA(siRNA)质粒EH-1和EH-2,以含非特异性siRNA编码序列的质粒(PCN)为阴性对照,用FuGENE HD转染原代培养的心肌细胞,通过半定量RT-PCR和Western blot法检测sEH的mRNA和蛋白的表达情况,筛选出抑制sEH效果最明显的表达质粒,命名为EH-R。采用浓度为10μmol.L-1ISO诱导心肌凋亡。分为正常对照组、ISO组、PCN+ISO组和EH-R+ISO组。利用EH-R下调心肌细胞sEH基因的表达,观察其对ISO诱导的心肌细胞凋亡的影响,流式细胞仪检测各组细胞凋亡的发生率,Western blot法检测各组Bcl-2和Bax蛋白的表达变化。结果:质粒EH-R组心肌细胞sEH的mRNA和蛋白质表达较其余组明显降低,差异有统计学意义(P<0.01)。与正常对照组相比,应用ISO各组,心肌细胞凋亡率明显升高,心肌细胞的Bax表达升高,而Bcl-2表达下降(P<0.01)。然而EH-R+ISO组与ISO组和PCN+ISO组相比心肌细胞凋亡率明显降低;心肌Bax表达降低,Bcl-2表达升高(P<0.01)。结论:构建了特异性小干扰RNA质粒,利用RNAi技术成功下调了原代培养的心肌细胞中sEH的表达,从而增加了抑凋亡基因Bcl-2的表达,减轻了ISO诱导心肌细胞的凋亡。为进一步进行心肌细胞的RNA干扰研究奠定了基础。

Objective：To construct the Soluble Epoxide Hydrolase（sEH） gene-specific recombinant Plasmids,and selectively knockdown the expression of sEH in mice cardiomyocytes by RNA interference（RNAi）,and to select the plasmids having the best silence effect to sEH.To investigate the effects of inhibition of sEH on apoptosis-related genes and change of Isoproterenol（ISO） induced cardiomyocytes apoptosis.Method：Two pairs of siRNAs that target at sEH gene were designed,and to construct the siRNA expression plasmids EH-1 and EH-2 specific to sEH gene.There are plasmids carrying a nonspecific siRNA coding sequence（PCN） as the negative control group.Then the plasmids were transfected into primary cultured cardiomyocytes by FuGENE HD.The mRNA and protein expressions of sEH were analyzed by RT-PCR and Western blot respectively.And to select the plasmids having the best silence effect to sEH,renamed EH-R.ISO at concentration of 10 μmol·L-1 induced cardiomyocytes apoptosis.Cardiomyocytes were divided into four groups： control group,ISO group,PCN＋ISO group and EH-R＋ISO group.Inhibition of sEH by EH-R plasmid.Rates of cardiomyocytes apoptosis were evaluated by flow cytometery,the protein expression levels of Bcl-2 and Bax were detected by Western blot.Result：The expressions of mRNA and protein of sEH in EH-R group were significantly decreased than that of other group（P0.01）.Applied ISO groups compare with C group,rates of cardiomyocytes apoptosis obviously increased,the expression levels of Bax increased,and the expression levels of Bcl-2 decreased（P0.01）.However EH-R＋ISO group compare with ISO group and PCN＋ISO group,the expression levels of Bax decreased,and the expression levels of Bcl-2 increased（P0.01）,and the rate of cardiomyocytes apoptosis was obviously decreased.Conclusion：Reco-mbinant plasmids have been constructed,and RNA interference can selectively knockdown sEH expression in cultured cardiomyocytes.Thereby it increased the expression of Bcl-2.Ameliorate cardiomyocytes apoptosis induced by ISO.This lays a foundation for further researches of RNAi on cardiomyocytes.