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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 a RNA interference plasmid containing soluble epoxide hydrolase (sEH) gene and selectively down-regulate the expression of sEH in mouse cardiomyocytes, and to screen out the expression plasmids that have the most obvious inhibitory effect on sEH. Adrenaline (ISO) - induced apoptosis of cardiomyocytes and the effects of related genes. Methods: Two specific small interfering RNA (siRNA) plasmids, EH-1 and EH-2, targeting sEH gene were constructed. Plasmid with non-specific siRNA coding sequence (PCN) was used as a negative control. FuGENE HD was used to transfect primary Cultured cardiomyocytes. The expression of sEH mRNA and protein was detected by semi-quantitative RT-PCR and Western blot, and the expression plasmid with the most obvious inhibitory effect on sEH was screened and named as EH-R. Cardiomyocytes apoptosis was induced by 10μmol.L-1ISO. Divided into normal control group, ISO group, PCN + ISO group and EH-R + ISO group. EH-R was used to down-regulate the expression of sEH gene in cardiomyocytes and to observe its effect on ISO-induced cardiomyocyte apoptosis. Flow cytometry was used to detect the apoptosis rate of each group. Western blot was used to detect the expression of Bcl-2 and Bax Changes in protein expression. Results: The mRNA and protein expression of sEH in EH-R group were significantly lower than those in the other groups (P <0.01). Compared with the normal control group, the apoptosis rate of cardiomyocytes was significantly increased, the expression of Bax increased, and the expression of Bcl-2 decreased in cardiomyocytes (P <0.01). However, the apoptosis rate of cardiomyocytes in EH-R + ISO group was significantly lower than that in ISO group and PCN + ISO group. The expression of Bax and Bcl-2 in myocardium were decreased (P <0.01). CONCLUSION: The specific small interfering RNA plasmid was constructed and the expression of sEH in primary cultured cardiomyocytes was successfully downregulated by using RNAi technology, which increased the expression of Bcl-2, and decreased the apoptosis of cardiomyocytes induced by ISO . Which laid the foundation for the further study of cardiac myocyte RNA interference.