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目的:观察缺血后处理中心肌细胞内源性细胞色素P450表氧化酶2J3/环氧二十碳三烯酸系统(CYP2J3/EETs)对心肌细胞凋亡的调节作用机制。方法:取Wistar乳鼠(12-24 h)心脏做原代心肌细胞培养。实验分为7组:对照组、缺氧/复氧组、缺氧后处理组、CYP2J3转染组、空质粒组、6-(2-炔丙基氧苯基)己酸(PPOH,一种CYP2J3抑制剂)组、二甲基亚砜(DMSO)溶剂组。除对照组外,其它各组均进行缺氧/复氧处理。用MTT法检测心肌细胞活力,高压液相色谱法检测心肌细胞培养液中11,12-EET浓度,Western blotting检测心肌细胞中caspase-3蛋白的表达,caspase-3活性检测试剂盒检测caspase-3蛋白的活性。结果:缺氧后处理组心肌细胞活力和11,12-EET浓度明显高于缺氧/复氧组(P<0.01),CYP2J3转染组比后处理组明显增高(P<0.01),PPOH组比后处理组明显下降(P<0.01)。后处理组caspase-3蛋白的表达及活性低于缺氧/复氧组(P<0.01),CYP2J3转染组caspase-3蛋白表达及活性低于后处理(P<0.01),而PPOH组caspase-3蛋白表达及活性高于缺氧后处理组(P<0.01)。结论:在缺氧后处理中CYP2J3/EETs可能通过抑制caspase-3蛋白的表达及活性来影响细胞凋亡,从而对心肌起保护作用。
AIM: To investigate the regulatory mechanism of cardiomyocyte apoptosis induced by endogenous cytochrome P450 epoxidase 2J3 / epi-eicosatetraenoic acid system (CYP2J3 / EETs) in ischemic postconditioning cardiomyocytes. Methods: The heart of Wistar suckling rats (12-24 h) was used to culture primary cardiomyocytes. The experiment was divided into 7 groups: control group, hypoxia / reoxygenation group, hypoxia postconditioning group, CYP2J3 transfection group, empty plasmid group, 6- (2- propargyloxyphenyl) hexanoic acid CYP2J3 inhibitor) group, dimethyl sulfoxide (DMSO) solvent group. In addition to the control group, the other groups were hypoxia / reoxygenation treatment. The cardiomyocyte activity was measured by MTT assay. The concentration of 11,12-EET in cardiomyocytes was detected by high pressure liquid chromatography. The expression of caspase-3 protein was detected by Western blotting. Caspase-3 activity was detected by caspase-3 activity assay kit Protein activity. Results: Compared with the hypoxia / reoxygenation group, the viability and the 11,12-EET concentration of cardiomyocytes in hypoxia-treated group were significantly increased (P <0.01), and were significantly increased in CYP2J3-transfected group Than the post-treatment group decreased significantly (P <0.01). The expression and activity of caspase-3 protein in the aftertreatment group were lower than those in the hypoxia / reoxygenation group (P <0.01), and the expression and activity of caspase-3 protein in CYP2J3 transfected group was lower than that in the aftertreatment group (P <0.01) -3 protein expression and activity than hypoxia aftertreatment group (P <0.01). CONCLUSIONS: CYP2J3 / EETs may play a protective role in cardiomyocytes by inhibiting the expression and activity of caspase-3 protein during hypoxia postconditioning.