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目的探讨促红细胞生成素(EPO)对缺氧复氧(H/R)心肌细胞是否具有保护作用及其机制。方法以乳鼠心肌细胞株(H9C2细胞)为研究对象,将其分为对照组、H/R组及EPO干预组。其中对照组常规培养;H/R组缺氧2 h后复氧24 h;EPO干预组在缺氧2 h后加入不同浓度的EPO(5、10、20 IU/ml)处理,然后复氧24 h。培养结束后分别检测各组细胞上清液中乳酸脱氢酶(LDH)浓度,使用MTS法检测细胞存活率,使用Western blot检测细胞内Cleaved Caspase-3表达变化,使用线粒体和胞浆蛋白制备试剂盒分离线粒体和胞浆蛋白,使用Western blot分别检测线粒体和胞浆内Omi/HtrA2蛋白表达变化。结果与对照组比较,H/R组细胞存活率明显下降,细胞上清液LDH释放明显增加,细胞内Cleaved Caspase-3表达增强,胞浆内Omi/HtrA2蛋白表达增强,差异均有统计学意义(P<0.05);而与H/R组相比,EPO干预组细胞存活率上升,细胞上清液LDH释放降低,Cleaved Caspase-3表达减弱,胞浆内Omi/HtrA2蛋白表达减少,差异均有统计学意义(P<0.05),但各指标均未恢复至对照组水平。结论 H/R可诱发心肌细胞凋亡,EPO可减轻H/R诱导的心肌细胞凋亡,其机制可能为抑制线粒体内Omi/HtrA2蛋白的转位,从而抑制Caspases通路激活有关。
Objective To investigate whether erythropoietin (EPO) has a protective effect on hypoxia-reoxygenation (H / R) cardiomyocytes and its mechanism. Methods The neonatal rat cardiomyocytes (H9C2 cells) were divided into control group, H / R group and EPO intervention group. The rats in the control group were cultured routinely for 24 hours and the rats in the H / R group were reoxygenated for 24 hours after hypoxia for 2 hours. The rats in the EPO group were treated with different concentrations of EPO (5,10,20 IU / ml) 2 hours after hypoxia, then reoxygenated 24 h After culture, the concentrations of lactate dehydrogenase (LDH) in the supernatant of each group were detected, the cell viability was detected by MTS method, the expression of Cleaved Caspase-3 was detected by Western blot, and mitochondria and cytosolic protein were used to prepare the reagent The mitochondria and cytoplasmic proteins were isolated and the expression of Omi / HtrA2 in mitochondria and cytoplasm were detected by Western blot. Results Compared with the control group, the cell viability was significantly decreased in H / R group, the release of LDH in the supernatant was significantly increased, the expression of Cleaved Caspase-3 was increased and the expression of Omi / HtrA2 in cytoplasm was increased (P <0.05). Compared with H / R group, the survival rate of EPO group increased, the release of LDH decreased, the expression of Cleaved Caspase-3 decreased and the expression of Omi / HtrA2 protein decreased There was statistical significance (P <0.05), but all the indexes did not recover to the control group. Conclusions H / R can induce cardiomyocyte apoptosis. EPO can reduce H / R-induced apoptosis in cardiomyocytes. The mechanism may be related to the inhibition of the translocation of Omi / HtrA2 protein in mitochondria and the inhibition of the activation of Caspases pathway.