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目的:研究蛋白激酶B(Akt)在促红细胞生成素(EPO)增强慢性缺氧环境中心肌细胞线粒体生物合成中的作用机制。方法:采用H9c2心肌细胞,将其于缺氧环境下培养7d(94%N2,1%O2,5%CO2),建立心肌细胞慢性缺氧模型。将心肌细胞根据不同处理分为缺氧对照组(HC组),20U/ml重组人EPO(rhEPO)处理缺氧组(HE组)和20U/ml rhEPO+100nmol/ml wortmannin特异性阻断Akt缺氧组(HW组)。以电镜观察线粒体超微结构变化,计算线粒体的体密度(Vv)、数密度(Nv);荧光探针观察检测线粒体数量变化;RT-PCR检测线粒体DNA相对拷贝数变化;Western blot检测Akt总蛋白表达及磷酸化水平变化。结果:在rhEPO作用后,Akt磷酸化显著增强(P<0.05),电镜观察显示线粒体Vv及Nv均显著升高(均P<0.05),线粒体数量及其DNA相对拷贝数也显著增加(P<0.05);采用wortmannin特异性阻断Akt使其磷酸化水平显著降低(P<0.05),同时使线粒体Vv、Nv、线粒体数量及其DNA相对拷贝数减少(均P<0.05)。结论:EPO通过促进Akt磷酸化进而增强慢性缺氧心肌细胞线粒体生物合成。
AIM: To investigate the role of Akt in the mitochondrial biogenesis of cardiomyocytes induced by erythropoietin (EPO) in chronic hypoxic environment. Methods: H9c2 cardiomyocytes were cultured in hypoxia for 7 days (94% N2, 1% O2, 5% CO2) to establish a model of cardiomyocyte chronic hypoxia. Myocardial cells were divided into hypoxic control group (HC group), 20U / ml recombinant human EPO (rhEPO) -treated hypoxia group (HE group) and 20U / ml rhEPO + 100nmol / Oxygen group (HW group). The ultrastructural changes of mitochondria were observed by electron microscopy. The density (Vv) and number density (Nv) of mitochondria were calculated. The number of mitochondria was detected by fluorescent probe. The relative copy number of mitochondrial DNA was detected by RT- Expression and phosphorylation level changes. Results: The phosphorylation of Akt was significantly enhanced after rhEPO treatment (P <0.05). The results of electron microscopy showed that the Vv and Nv of mitochondria increased significantly (both P <0.05) and the number of mitochondria and the relative copy number of DNA significantly increased (P < 0.05). The specific phosphorylation of Akt by wortmannin significantly decreased the phosphorylation level of mitochondrial Vv, Nv, mitochondria and relative DNA copy number (P <0.05). Conclusion: EPO enhances mitochondrial biogenesis in chronic hypoxic cardiomyocytes by promoting Akt phosphorylation.