急性缺氧肺动脉平滑肌细胞线粒体活性氧变化的观察

发布时间：2018-03-18 00:24

本文选题：活性氧　切入点：缺氧　出处：《中国病理生理杂志》2014年11期 　论文类型：期刊论文

【摘要】：目的:观察急性缺氧时大鼠肺动脉平滑肌细胞(PASMCs)线粒体活性氧(ROS)的变化。方法:分离并培养大鼠PASMCs,常氧(35℃、5%CO2、21%O2、74%N2)和急性缺氧(35℃、5%CO2、1%O2、94%N2)条件下,利用分子探针chloromethyl dichlorodihydrofluorescein diacetate(CM-H2DCF/DA)和Redox Sensor Red CC-1通过激光共聚焦显微镜来检测细胞内ROS的生成量;直接分离线粒体,用线粒体电子传递链(ETC)复合物抑制剂通过荧光分光光度计检测线粒体ROS及其生成的具体位点。结果:急性缺氧细胞内ROS生成明显增加,其中缺氧组H2O2较常氧组增加3.35倍(P0.01),而H2O2及O-·2较常氧组增加1.61倍(P0.01)。与缺氧组比,用线粒体ETC复合物I抑制剂MPP、复合物II抑制剂NPA和TTFA及复合物III前泛半醌位点抑制剂myxothiazol都能显著降低缺氧时PASMCs胞内ROS的生成量(分别降低60%、73%、75%和61%,P0.01);而复合物III后泛半醌位点抑制剂antimycin A及复合物IV抑制剂Na N3对ROS的生成无明显影响(升高13%和9.1%,P0.05)。直接检测线粒体ROS与测定细胞内ROS结果一致。结论:急性缺氧PASMCs线粒体ROS(主要是H2O2)的生成量明显增加;其生成位点主要是线粒体ETC复合物Ⅰ、Ⅱ及III前泛半醌位点,而与复合物III后泛半醌位点和IV位点无明显关系。
[Abstract]:Objective: To observe the effect of acute hypoxia on rat pulmonary artery smooth muscle cells (PASMCs) of mitochondrial reactive oxygen species (ROS) changes. Methods: isolated and cultured rat PASMCs, normoxia (at 35 5%CO2,21%O2,74%N2) and acute hypoxia (35 C, 5%CO2,1%O2,94%N2) under the condition of using molecular probe chloromethyl dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) and Redox Sensor Red CC-1 by laser scanning confocal microscope to detect intracellular ROS production; mitochondria isolated directly, with the mitochondrial electron transfer chain (ETC) inhibitor complexes by fluorescence spectrophotometer to detect specific sites of mitochondrial ROS and its formation. Results: acute hypoxia significantly increased intracellular ROS generation, which is often H2O2 hypoxia group oxygen group increased 3.35 times (P0.01), H2O2 and O- 2 compared with normoxia group increased 1.61 times (P0.01) and hypoxia group. Compared with mitochondrial ETC complex I inhibitor MPP, complex II inhibition Agent NPA and TTFA and III complexes before ubisemiquinone site inhibitor myxothiazol can significantly reduce the amount of hypoxia generated PASMCs intracellular ROS (respectively reduced 60%, 73%, 75% and 61%, P0.01); and III complex after ubisemiquinone site inhibitor antimycin A and complex IV inhibitor Na N3 on ROS generation has no obvious effect (increased by 13% and 9.1%, P0.05). The direct detection of mitochondrial ROS and determination of intracellular ROS results. Conclusion: acute hypoxia PASMCs mitochondrial ROS (H2O2) production increased significantly; the main site is ETC mitochondrial complexes I, II and III before ubisemiquinone sites, and with the complex III ubisemiquinone loci and IV loci showed no significant relationship.

【作者单位】：宁夏人民医院总院呼吸内科;宁夏人民医院心血管内科;第四军医大学西京医院呼吸内科;
【基金】：国家自然科学基金资助项目(No.81160011) 宁夏回族自治区科技攻关资助项目(宁陕合作)(No.2012ZYH225)
【分类号】：R364

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