Diazoxide对缺氧大鼠肺动脉平滑肌细胞内氧自由基的变化及细胞增殖的作用  

Effect of diazoxide on change of H_2O_2 in rat pulmonary artery smooth muscle cells and proliferation of hypoxic rat pulmonary artery smooth muscle cells

作　　者：胡红玲[1] 汪涛[1] 张珍祥[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所,湖北武汉430030

出　　处：《中国病理生理杂志》2007年第10期2002-2006,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30370623)

摘　　要：目的:研究线粒体膜上ATP敏感钾通道(MitoKATP)开放剂diazoxide和线粒体膜电位(ΔΨm)在缺氧导致的大鼠肺动脉平滑肌细胞内氧自由基的变化及细胞增殖/凋亡失衡中的作用,探索缺氧性肺动脉重建和肺动脉高压形成的发生机制。方法: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。将样本分为6组:①正常对照组;②线粒体膜上ATP敏感钾通道(MitoKATP)开放剂diazoxide组;③MitoKATP阻断剂5-HD组;④慢性缺氧(CH)组;⑤CH+diazoxide组;⑥CH+5-HD组。利用激光共焦显微镜(Leica SP-1 USA)成像检测线粒体膜电位,荧光染色检测细胞内氧自由基含量,流式细胞仪检测细胞周期和MTT法检测细胞增殖情况。结果:①Diazoxide作用24 h后,R-123荧光强度明显强于正常对照组,线粒体膜电位去极化,细胞内氧自由基含量明显高于正常对照组,细胞增殖明显多于正常对照组、凋亡少于正常对照组,P<0.05;而5-HD作用24 h后,上述指标与正常对照组相比较,均无显著差异,P>0.05;②慢性缺氧24 h,结果与di-azoxide组相似,R-123荧光强度明显强于正常对照组,线粒体膜电位去极化,细胞内氧自由基含量明显高于正常对照组,细胞增殖明显多于正常对照组、凋亡少于正常对照组,P<0.05;CH+diazoxide组,R-123荧光强度和线粒体膜电位去极化明显强于缺氧组,细胞内氧自由基含量明显高于缺氧组,细胞增殖明显多于缺氧组、凋亡少于缺氧组,P<0.05;CH+5-HD组,R-123荧光强度和线粒体膜电位去极化明显弱于缺氧组,细胞内氧自由基含量明显低于缺氧组,细胞增殖明显少于缺氧组、凋亡多于缺氧组,P<0.05;R-123荧光强度、MTT的A值与细胞内氧自由基含量均呈显著正相关;凋亡细胞百分比与细胞内氧自由基含量呈显著负相关。结论:本实验结果显示,diazox-ide能够通过开放线粒体膜上ATP敏感的钾通道,引起ΔΨm去极化,ΔΨm的变化影响细胞内AIM： To investigate the contribution of diazoxide, an opener of mitochondrial ATP- sensitive K^＋ channel （MitOKATP）, and mitochondrial membrane potential （△ψm） to change of H2O2 in rat pulmonary artery smooth muscle cells （PASMCs） and to unbalance between cell proliferation and apoptosis of PASMCs induced by hypoxia. METHODS ： The rat PASMCs were isolated from fresh normal lung tissues and cultured, which were divided into 6 groups, as follows： ① control group; ② diazoxide group; ③ 5 -HD group; ④chronic hypoxia group; ⑤ chronic hypoxia ＋ diazoxide group ; ⑥chronic hypoxia ＋ 5 - HD group. The relative change in mitochondrial potential was detected with rhodamine fluorescence （R- 123） technique. The level of H2O2 in rat PASMCs was detected with chemiluminescence method. The proliferation of rat PASMCs was examined by cell cycle analysis and MTr colorimetric assay. RESULTS： After exposed to diazoxide for 24 h, the intensity of R - 123 fluorescence, the level of H2O2 and the A value in normoxic rat PASMCs were significantly increased, and the apoptosis of rat PASMCs was significantly decreased as compared with control group （P 〈 0. 05）. However, there were no significant changes in these tests after the rat PASMCs had been exposed to 5 - HD for 24 h. Chronic hypoxia or chronic hypoxia ＋ diazoxide markedly increased the intensity of R - 123 fluorescence, the level of H2 O2 and the A value in rat PASMCs, and also markedly decreased the apoptosis of rat PASMCs as compared with control group （ P 〈 0. 05 ）, and these changes were more significant in chronic hypoxia ＋ diazoxide group than those in chronic hypoxia group （ P 〈 0. 05 ）. 5 - HD partly weakened the effect of hypoxia on the intensity of R - 123 fluorescence, the level of H2O2 , the A value and the apoptosis of rat PASMCs （ P 〈 0. 05 ）. Significant and positive correlations were found between the intracellular H2O2 and the R - 123 fluorescence or the A value. Significant and negative correla

关 键 词：肺动脉 平滑肌细胞 缺氧 线粒体膜电位 线粒体ATP敏感性钾通道 活性氧 

分 类 号：R363[医药卫生—病理学]