摘要
为了探讨线粒体ATP敏感钾通道(mitochondrial ATP-sensitive K^+channel,mitoK_(ATP))和线粒体膜电位(ΔΨm)在细胞缺氧信号转导中的作用以及对缺氧肺动脉平滑肌细胞中细胞色素C在细胞内的分布及细胞增殖的影响,本实验将人肺动脉平滑肌细胞进行常氧或24h缺氧培养,并将标本分为六组：(1)对照组；(2)mitoK_(ATP)开放剂diazoxide组；(3)mitoK_(ATP)阻断剂5-HD组：(4)24h缺氧组：(5)24h缺氧+diazoxide组；(6)24h缺氧+5．HD组。利用激光共聚焦显微镜成像法检测ΔΨm：线粒体/胞浆成分分离试剂盒(BioVision)分离线粒体和胞浆成分后,Western blot检测两者细胞色素C：Western blot检测细胞中caspase-9的蛋白表达量；MTT法及PI染色后流式细胞仪检测细胞增殖情况。结果显示：(1)diazoxide作用24h后,R-123荧光明显增强,胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞增殖明显增多、凋亡减少,与正常对照组相比较,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(P＞0．05)。(2)缺氧24h组,结果与diazoxide组相似,R-123荧光明显增强,胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞增殖明显增多、凋亡减少,与正常对照组相比较,均P＜0．05：24h缺氧+diazoxide组与缺氧组相比较,R-123荧光明显增强,胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞增殖明显增多、凋亡减少(P＜0．05)；而24h缺氧+5-HD组与缺氧组比较,R-123荧光明显降低,胞浆细胞色素C与线粒体细胞色素C的比值明显升高,caspase-9的蛋白表达显著增加,细胞增殖明显减少、凋亡增多(P＜0．05)。上述实验结果提示,缺氧可以引起mitoK_(ATP)的开放以及ΔΨm的去极化,并进而抑制细胞色素C从线粒体释放到胞浆,抑制线粒体凋亡途径,从而参与并影响肺动脉高压
The objective of this paper was to investigate the contribution of mitochondrial ATP-sensitive K^＋ channel （mito KATP） and mitochondrial membrane potential （△ψm） to the distribution of cytochrome C in human pulmonary arterial smooth muscle cells （HPASMCs） and to the proliferation of HPASMCs induced by hypoxia. HPASMCs were divided into 6 groups, as following： （1） control group： cultured under normoxia; （2） diazoxide group： cultured in normoxia with diazoxide, an opener of mitoKATP; （3） 5-HD group： cultured in normoxia with 5-hydroxydecanoate （5-HD）, an antagonist of mitoKATP; （4） 24-hour hypoxia group： cultured in hypoxia for 24 h; （5） 24-hour hypoxia ＋ diazoxide group： cultured in hypoxia with diazoxide for 24 h; （6） 24-hour hypoxia ＋ 5-HD group： cultured in hypoxia with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with rhodamine fluorescence （R-123） technique. Western blot was used to detect the expression of cytochrome C protein in cell plasma and mitochondria, respectively. The expression of cell caspase-9 protein was determined with Western blot. The proliferation of HPASMCs was examined by cell cycle analysis and MTT colorimetric assay. The results were as following： after exposure to diazoxide for 24 h, the intensity of R-123 fluorescence in normoxic HPASMCs was significantly increased compared with that in the control group （P〈0.05）, but there was no significant change of the intensity of R-123 fluorescence after the HPASMCs had been exposed to 5-HD for 24 h; 24- hour hypoxia or 24-hour hypoxia ＋ diazoxide could markedly increase the intensity of R-123 fluorescence in HPASMCs compared with normoxia （P〈0.05）, and the change was more significant in 24-hour hypoxia ＋ diazoxide group than that in 24-hour hypoxia group （P〈0.05）; 5-HD could weaken the effect of 24-hour hypoxia on the intensity of R-123 fluorescence. After exposure to diazoxide for 24 h, the ratio of the expression of cytosoli
出处
《生理学报》
CAS
CSCD
北大核心
2006年第3期262-268,共7页
Acta Physiologica Sinica
基金
This work was supported by the National Natural Science Foundation of China(No.30370623).
关键词
肺动脉平滑肌细胞
缺氧
线粒体膜电位
线粒体膜ATP敏感钾通道
细胞色素C
pulmonary arterial smooth muscle cell
hypoxia
mitochondrial membrane potential
mitochondrial ATP-sensitive potassium channel
cytochrome C
