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利用过氧化氢(H_2O_2)建立大鼠心肌细胞氧化损伤模型;观察心肌细胞氧化损伤过程中myocardin和核因子E2相关因子2(Nrf2)的表达变化并初步探讨myocardin对Nrf2的影响。通过转染质粒过表达目的基因,转染sh RNA质粒下调目的基因表达;通过磺酰罗丹明B(SRB)比色法检测细胞增殖,通过Real-time PCR检测mRNA的表达,通过Western blot检测蛋白的表达。结果显示200μmol/L H_2O_2孵育24 h为最佳H_2O_2氧化损伤条件;H_2O_2抑制myocardin mRNA及蛋白的表达,同时增加Nrf2 mRNA及蛋白的表达;过表达myocardin基因或者下调Nrf2基因后相对活细胞数较对照组明显减少,而下调myocardin基因或者上调Nrf2基因后相对活细胞数较对照组明显增多;过表达myocardin基因后检测到Nrf2 mRNA和蛋白表达出现明显下调,而下调myocardin基因后检测到Nrf2 mRNA和蛋白表达明显上调。因此推断myocardin基因可能抑制细胞增殖,而Nrf2基因可能促进细胞增殖;H_2O_2造成大鼠心肌细胞氧化损伤过程中激活Nrf2相关抗氧化损伤信号途径,其机制可能是通过下调myocardin的表达而实现的。
Oxidative damage of cardiomyocytes was induced by hydrogen peroxide (H_2O_2). The changes of myocardin and nuclear factor 2 (Nrf2) expression in cardiomyocytes during oxidative injury were observed and the effect of myocardin on Nrf2 was also investigated. The transfected plasmid was transfected with sh RNA plasmid to down-regulate the expression of the target gene. The cell proliferation was detected by SRB colorimetric assay. The expression of mRNA was detected by Real-time PCR. The protein was detected by Western blot expression. H 2 O 2 inhibited the expression of myocardin mRNA and protein and increased the expression of Nrf2 mRNA and protein. The number of viable cells after overexpression of myocardin gene or down-regulation of Nrf2 gene was higher than that of control Group decreased significantly, and the number of viable cells after the down-regulation of myocardin gene or up-regulation of Nrf2 gene was significantly higher than that of the control group. The expression of Nrf2 mRNA and protein was significantly down-regulated after overexpression of myocardin gene, but downregulation of the myocardin gene detected Nrf2 mRNA and protein The expression was significantly increased. Therefore, it is concluded that myocardin gene may inhibit cell proliferation and Nrf2 may promote cell proliferation. H 2 O 2 may induce the oxidative damage of cardiomyocytes induced by N 2 O 2, and its mechanism may be through down-regulating the expression of myocardin.