线粒体膜电位对缺氧大鼠肺动脉平滑肌细胞内细胞色素C分布及细胞增殖的影响  

Effect of Mitochondrial Membrane Potential on the Distribution of Cytochrome C and the Proliferation of Hypoxic Rat Pulmonary Artery Smooth Muscle Cells

作　　者：胡红玲[1] 张珍祥[1] 汪涛[1] 赵建平[1] 徐永健[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院呼吸疾病研究室,武汉430030

出　　处：《华中科技大学学报（医学版）》2008年第2期166-169,280,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.30370623)

摘　　要：目的从线粒体ATP敏感性钾通道(MitoKATP)入手,研究在缺氧影响大鼠肺动脉平滑肌细胞内细胞色素C的分布及其引起细胞增殖变化中线粒体膜电位(Δψm)的作用。方法获取大鼠正常肺组织,分离出肺动脉平滑肌细胞(PASMCs)进行常氧或慢性缺氧培养。将标本分为6组:①正常对照组;②MitoKATP开放剂Diazoxide组;③MitoKATP阻断剂5-hydroxydecanoate(5-HD)组;④慢性缺氧(CH)组;⑤CH+Diazoxide组;⑥CH+5-HD组。利用激光共聚焦显微镜成像检测线粒体膜电位,Western blot法检测线粒体和细胞质中细胞色素C蛋白含量,流式细胞仪检测细胞周期,MTT法检测细胞增殖情况。结果①Diazoxide作用24 h后,细胞R-123荧光强度明显增强,线粒体膜电位去极化,细胞胞质与线粒体细胞色素C的比值明显降低,细胞明显增殖,凋亡减少,与正常对照组比较均P<0.05;而5-HD作用24h与正常对照组比较,上述指标无明显变化,均P>0.05;②慢性缺氧24 h,结果与Diazoxide组相似,各项指标与正常对照组比较均P<0.05;CH+Diazoxide组与CH组比较,细胞R-123荧光强度明显增强,线粒体膜电位去极化,细胞胞质与线粒体细胞色素C的比值明显降低,细胞明显增殖,凋亡减少,与CH组比较均P<0.05;CH+5-HD组与CH组比较,细胞R-123荧光强度明显减弱,线粒体膜电位部分消失,细胞胞质与线粒体细胞色素C的比值明显升高,细胞增殖减少,凋亡增多,与CH组比较均P<0.05。结论缺氧可以引起MitoKATP的开放以及线粒体膜电位去极化,进而抑制了细胞色素C从细胞线粒体释放到细胞质,抑制线粒体凋亡途径,从而参与并影响了肺动脉高压的发生发展。Objective To investigate the contribution of mitochondrial membrane potential （△ψm） and mitochondrial ATP- sensitive K^＋ channel （MitoKATP） to the distribution of cytochrome C and the proliferation of rat pulmonary artery smooth muscle cells （PASMCs） induced by hypoxia. Methods The rat PASMCs were isolated from fresh lung of normal rats and cultured, and then were divided into 6 groups as follow： control group, cultured under normoxia; diazoxide group, cultured under normoxia with diazoxide, an opener of MitoKATP. for 24 h; 5-HD group, cultured under normoxia with 5-hydroxydecanoate （5-HD）, an antagonist of MitoKATP for 24 h; chronic hypoxia group, cultured under hypoxia for 24 h; chronic hypoxia＋diazoxide group; chronic hypoxia＋ 5-HD group. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence （R-123） technique. Western blot technique was used to detect the expression of cytochrome C protein in cell plasma and mitochondria respectively. The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay. Results The intensity of R-123 fluorescence and the A value of the rat PASMCs in diazoxide group were significantly increased, the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria and the apoptosis of rat PASMCs were significantly decreased as compared with control group （all P〈0.05）. But there were no significant changes in these tests of 5-HD group （all P〉0.05）. Chronic hypoxia or chronic hypoxia＋ diazoxide could markedly increase the intensity of R-123 fluorescence and the A value in rat PASMC as compared with control group （P〈0.05）, and could markedly decrease the rate of the expression of cytochrome C protein in cell plasma to that in cell mitochondria and the apoptosis of rat PASMCs, all these changes were more significant in chronic hypoxia＋ diazoxide group than in chronic hypoxia group （P〈0. 05）. 5-HD could weaken all these effect of hypoxia （

关 键 词：肺动脉平滑肌细胞 缺氧 钾通道 线粒体膜电位 细胞色素C 

分 类 号：R563[医药卫生—呼吸系统]