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线粒体ATP敏感钾通道对大鼠肺动脉平滑肌细胞低氧诱导因子--1#alpha#表达及细胞增殖的作用

赵建平, 郭治, 周志刚, 陈俊, 胡红玲, 汪涛, 张珍祥

华中科技大学同济医学院附属同济医院呼吸科.湖北,武汉 430030

摘要

该文旨在探讨线粒体ATP敏感钾(mitochondrial ATP--sensitive K~(+),MitoK_(ATP))通道对大鼠肺动脉平滑肌细胞低氧诱导因子--1#alpha#(hypoxia inducible factor--1#alpha#,HIF--1#alpha#)表达和细胞增殖的影响。原代培养大鼠肺动脉平滑肌细胞,分为常氧对照组、常氧+diazoxide(MitoK_(ATP)通道的选择性开放剂)组、常氧+5--hydroxydecanoate(5--HD,MitoK_(ATP)通道的选择性阻断剂)组、低氧对照组、低氧+diazoxide组、低氧+5--HD组,共6组,分别应用罗丹明123荧光技术检测各组大鼠肺动脉平滑肌细胞的线粒体膜电位,免疫组化检测HIF--1#alpha#的表达及酶联免疫检测仪检测细胞增殖的变化。结果显示,常氧+diazoxide组与常氧对照组比较,罗丹明123荧光、HIF--1#alpha#表达及细胞增殖明显增强({sl P}＜0.05);低氧+diazoxide组与低氧对照组比较,罗丹明123荧光、HIF--1#alpha#表达及细胞增殖明显增强({sl P}＜0.05);常氧+5--HD组与常氧对照组比较,罗丹明123荧光、HIF--1#alpha#表达、细胞增殖没有明显变化({sl P}＞0.05);但低氧+5--HD组与低氧对照组比较,罗丹明123荧光明显减弱、HIF--1#alpha#表达及细胞增殖有所减弱({sl P}＜0.05)。结果提示:MitoK_(ATP)通道的开放能引起大鼠肺动脉平滑肌细胞线粒体膜去极化,并可以促进HIF--1#alpha#的表达及细胞增殖。

关键词： 低氧; 低氧诱导因子-1#alpha#; 线粒体膜电位; 平滑肌

Effect of opening of mitochondrial ATP--sensitive K~(+) channels on the expression of hypoxia inducible factor--1#alpha# and cell proliferation in pulmonary arterial smooth muscle cells of rats

Zhao Jianping, Guo Zhi, Zhou Zhigang, Chen Jun, Hu Hongling, Wang Tao, Zhang Zhenxiang

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

Abstract

The objective of this paper was to investigate the effect of mitochondrial ATP-sensitive K~(+) (MitoK_(ATP) channel on the expression of HIF-1#alpha# and cell proliferation in pulmonary arterial smooth muscle cells (PASMCs) of rats. Cultured PASMCs were divided into six groups as follows: (1) normoxia group: cultured under normoxia; (2) normoxia + diazoxide group: cultured in normoxia with diazoxide, an opener of MitoK_(ATP); (3) normoxia + 5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of MitoK_(ATP); (4) hypoxia group: cultured under hypoxia (37℃, 5% O_(2), 5% CO_(2), 90% N_(2)) for 24 h; (5) hypoxia + diazoxide group, cultured under hypoxia (37℃, 5% O_(2), 5% CO_(2), 90% N_(2)) with diazoxide for 24 h; (6) hypoxia + 5-HD group, cultured under hypoxia (37℃, 5% O_(2), 5% CO_(2), 90% N_(2)) with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with Rhodamine fluorescence (R-123) technique. Immunohistochemical method was used to trace the expression of HIF-1#alpha#. The proliferation of PASMCs was examined by MTT colorimetric assay. The results were as follows: The intensity of R-123 fluorescence in normoxia + diazoxide group was significantly increased as compared with that in normoxia group (P<0.05), and the intensity of R-123 fluorescence in hypoxia + diazoxide group was also significantly increased as compared with that in hypoxia group (P<0.05). 24-hour hypoxia or 24-hour hypoxia + diazoxide markedly increased the intensity of R-123 fluorescence in PASMCs as compared with normoxia (P<0.05), and the change was more prominant in hypoxia + diazoxide group than that of hypoxia group (P<0.05). There was no significant difference in the intensity of R-123 fluorescence between normoxia group and normoxia + 5-HD group (P>0.05). However, 5-HD weakened the effect of 24-hour hypoxia on the intensity of R-123 fluorescence. The intensity of R-123 fluorescence in hypoxia + 5-HD group was significantly decreased as compared with that in hypoxia group (P<0.05). After exposure to hypoxia or hypoxia + diazoxide for 24 h, the expression of HIF-1#alpha# and the proliferation of PASMCs were significantly increased as compared with that in normoxia or normoxia + diazoxide group (P<0.05), and the change was more significant in hypoxia + diazoxide group than that in hypoxia group (P<0.05). There was no significant difference in the expression of HIF-1#alpha# and the proliferation of PASMCs between normoxia group and normoxia + 5-HD group (P>0.05). Since 5-HD could weaken the effect of 24-hour hypoxia, the expression of HIF-1#alpha# and the proliferation of PASMCs in hypoxia + 5-HD group were significantly decreased as compared with that in hypoxia group (P<0.05). All these results suggest that the opening of MitoK_(ATP) followed by a depolarization of mitochondrial membrane might contribute to the increase of the expression of HIF-1#alpha# and the proliferation of PASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension.

Key words: Hypoxia;hypoxia inducible factor-1#alpha#;Mitochondrial membrane potential;smooth muscle

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引用本文：

赵建平, 郭治, 周志刚, 陈俊, 胡红玲, 汪涛, 张珍祥. 线粒体ATP敏感钾通道对大鼠肺动脉平滑肌细胞低氧诱导因子--1#alpha#表达及细胞增殖的作用[J]. 生理学报 2007; 59 (2): 157-162.

Zhao Jianping, Guo Zhi, Zhou Zhigang, Chen Jun, Hu Hongling, Wang Tao, Zhang Zhenxiang. Effect of opening of mitochondrial ATP--sensitive K~(+) channels on the expression of hypoxia inducible factor--1#alpha# and cell proliferation in pulmonary arterial smooth muscle cells of rats. Acta Physiol Sin 2007; 59 (2): 157-162 (in Chinese with English abstract).