eNOS在EPO调节慢性缺氧心肌细胞线粒体生物合成中的作用机制研究  

The role of eNOS on the regulatory effects of EPO on mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia

作　　者：秦川[1] 陈林[1] 肖颖彬[1] 

机构地区：[1]第三军医大学新桥医院全军心血管外科研究所,重庆400037

出　　处：《重庆医学》2015年第16期2167-2169,2173,共4页Chongqing medicine

基　　金：国家自然科学基金资助项目(81100120)

摘　　要：目的探讨内皮型一氧化氮合酶(eNOS)在促红细胞生成素(EPO)调节慢性缺氧环境中心肌细胞线粒体生物合成中的作用及其机制。方法采用H9c2心肌细胞,将其于缺氧环境下培养7d(94%N2,5%O2),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),20 U/mL重组人EPO(rhEPO)处理缺氧组(HE)和20 U/mL rhEPO+eNOS shRNA干扰处理缺氧组(HR)。以荧光探针检测线粒体数量变化;RT-PCR检测线粒体DNA相对表达量;Western blot检测eNOS总蛋白表达及磷酸化水平(p-eNOS)变化。结果 rhEPO显著增强eNOS磷酸化水平,增加线粒体数量及其DNA相对拷贝数(P<0.05);而同时采用shRNA干扰eNOS后,HR组eNOS总蛋白表达及磷酸化水平较HE组降低(P<0.05),同时线粒体数量及其DNA相对拷贝数较HE组减少(P<0.05)。结论 eNOS的磷酸化激活是EPO增强慢性缺氧心肌细胞线粒体生物合成的重要信号转导机制。Objective To explore the role of endothelial nitric oxide synthase（eNOS） in the regulatory effects of erythropoie- tin （EPO） on mitochondiral biogenesis in eardiomyocytes exposed to chronic hypoxia. Methods H9c2 cardiomyocytes were cultured in the environment of hypoxia for 1 week（94 % N2,5 %02 ）, establishing the chronic hypoxic cardiomyocyte model. All the ceils were divided into 3 groups： HC（chronic hypoxic control）, HE[treated with chronic hypoxia and 20 U/mL recombinant human EPO （rhEPO） ]and HR（cells transfected with eNOS shRNA plasmid and treated with 20 U/mL rhEPO and chronic hypoxia）. Fluores- cent probe was used to detect the changes of mitochondial number. Mitochondial DNA （mtDNA） relative express level was assayed by RT-PCR. The expression and phosphorylation of eNOS protein were analyzed with Western blot. Results rhEPO significantly increased the phosphorylation of eNOS and elavated the mitochondialt number and mtDNA （P〈 0. 05）. shRNA interference on eNOS significantly blocked all the above changes induced by rhEPO （P〈0.05）. Conclusion Phosphory lation of eNOS is the important signalling pathway for the enhanced mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia by EPO.

关 键 词：促红细胞生成素 线粒体生物合成 慢性缺氧 心肌细胞 内皮型一氧化氮合酶 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]