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低氧条件下大鼠肺动脉平滑肌细胞中活性氧与低氧诱导因子--1#alpha#和细胞增殖的关系

赵建平, 周志刚, 胡红玲, 郭治, 汪涛, 甄国华, 张珍祥

华中科技大学同济医学院附属同济医院呼吸内科.湖北,武汉 430030

摘要

在低氧条件下，观察大鼠肺动脉平滑肌细胞（pulmonary arterial smooth muscle cells，PASMCs）中活性氧（reactive oxygen species，ROS）的变化，探讨ROS的变化是否通过调控低氧诱导因子--1#alpha#（hypoxia--inducible factor 1#alpha#, HIF--1#alpha#）的表达影响PASMCs的增殖。采用组织块法原代培养大鼠PASMCs，分成3组：常氧组（21％O_(2)，24h），低氧组（5％O_(2)，24h），低氧+Mn--TBAP组（5％O_(2)，24h，Mn--TBAP是一种ROS清除剂）。用激光共聚焦显微镜荧光染色法检测细胞内ROS的变化；用RT--PCR和免疫组织化学方法分别测定HIF--1#alpha# mRNA和蛋白的表达；用MTT法检测细胞增殖程度。结果显示：（1）低氧组PASMCs内ROS水平明显高于常氧组（{sl P}<0.05），低氧+Mn--TBAP组ROS水平明显低于低氧组（{sl P}<0.05），但仍高于常氧组（{sl P}<0.05）；（2）低氧组及低氧+Mn--TBAP组的HIF--1#alpha# mRNA和蛋白表达均高于常氧组（{sl P}<0.05），且低氧组表达高于低氧+Mn--TBAP组（{sl P}<0.05）；（3）低氧组细胞增殖明显高于常氧组和低氧+Mn--TBAP组（{sl P}<0.05），低氧+Mn--TBAP组细胞增殖高于常氧组（{sl P}<0.05）。结果表明：在低氧条件下大鼠PASMCs中ROS水平明显升高，RROS的变化能够调节HIF--1#alpha#的表达，进而影响平滑肌细胞的增殖，提示ROS可能在肺动脉高压的发病机制和低氧信号转导中具有重要作用。

关键词： 低氧; 平滑肌细胞; 活性氧; 低氧诱导因子-1; 增殖

The relationships among reactive oxygen species, hypoxia--inducible factor 1#alpha# and cell proliferation in rat pulmonary arterial smooth muscle cells under hypoxia

Zhao Jianping, Zhou Zhigang, Hu Hongling, Guo Zhi, Wang Tao, Zhen Guohua, Zhang Zhenxiang

Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.Wuhan 430030,Hubei

Abstract

The objectives of this paper were to observe the changes of reactive oxygen species （ROS） in rat pulmonary arterial smooth muscle cells （PASMCs） under hypoxic condition and to test if hypoxia-induced proliferation of PASMCs was mediated by ROS. PASMCs were divided into three groups： normal group, hypoxia group and hypoxia + Mn-TBAP （a ROS scavenger） group. The level of ROS was determined by a laser scanning confocal microscope. The expression of hypoxia-inducible factor 1#alpha#（HIF-1#alpha#） mRNA was detected by semi-quantitative reverse transcription PCR （RT-PCR）. HIF-1#alpha# protein was detected using immunohistochemical staining, and the proliferation of PASMCs was examined by MTT colorimetric assay. The results were as follows： （1） The level of ROS in hypoxia group was significantly increased as compared with that in the normal group （P〈0.05）. The level of ROS in hypoxia + Mn-TBAP group was significantly decreased as compared with that in hypoxia group （P〈0.05）, but was increased as compared with that in the normal group （P〈0.05）. （2） The expressions of HIF-1#alpha# mRNA and protein in hypoxia group and hypoxia + Mn-TBAP group were increased as compared with those in the normal group （P〈0.05）, and these changes were more significant in hypoxia group than those in hypoxia + Mn-TBAP group. （3） The proliferation of PASMCs in hypoxia group was more obvious than that in the normal group and hypoxia + Mn-TBAP group （P〈0.05）, and the proliferation of PASMCs in hypoxia + Mn-TBAP group was increased more significantly than that in the normal group （P〈0.05）. The results indicate that ROS is significantly increased in rat PASMCs under hypoxia, and that ROS affects the expression of HIF-1#alpha# and the proliferation of PASMCs under hypoxia. Therefore, ROS may play an important role in the pathogenesis of pulmonary hypertension and hypoxic signal transductions.

Key words: Hypoxia;Smooth muscle cells;Reactive oxygen species;hypoxia-inducible factor 1;Proliferation

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引用本文：

赵建平, 周志刚, 胡红玲, 郭治, 汪涛, 甄国华, 张珍祥. 低氧条件下大鼠肺动脉平滑肌细胞中活性氧与低氧诱导因子--1#alpha#和细胞增殖的关系[J]. 生理学报 2007; 59 (3): 319-324.

Zhao Jianping, Zhou Zhigang, Hu Hongling, Guo Zhi, Wang Tao, Zhen Guohua, Zhang Zhenxiang. The relationships among reactive oxygen species, hypoxia--inducible factor 1#alpha# and cell proliferation in rat pulmonary arterial smooth muscle cells under hypoxia. Acta Physiol Sin 2007; 59 (3): 319-324 (in Chinese with English abstract).