【摘要】 本文旨在探讨线粒体ATP敏感钾(mitochondrial ATP-sensitive K+,MitoKATP)通道对哮喘大鼠气道平滑肌细胞(airway smooth muscle cells,ASMCs)增殖的影响及其调控机制。36只Sprague-Dawley(SD)大鼠随机分为哮喘组(n=18)和正常组(n=18),哮喘组大鼠采用卵清蛋白(ovalbumin,OVA)致敏及激发的方法制备哮喘模型,正常组大鼠吸入等量生理盐水。取各组大鼠肺组织,分离出ASMCs进行培养,将样本分6组分别进行干预:(1)正常组;(2)正常+MitoKATP通道开放剂diazoxide组(diazoxide组);(3)正常+MitoKATP通道阻断剂5-HD组(5-HD组);(4)哮喘(asthma)组;(5)asthma+diazoxide组;(6)asthma+5-HD组。利用罗丹明(Rhodamine123,R-123)荧光染色、激光共聚焦显微镜成像检测线粒体膜电位(ΔΨm),DCFH-DA荧光染色法检测细胞内活性氧(reactive oxygen species,ROS)含量,RT-PCR检测核因子κB(nuclear factor-κB,NF-κB)mRNA的表达,流式细胞仪检测细胞凋亡和MTT法检测细胞增殖情况。结果显示:(1)Diazoxide组与正常组相比,R-123荧光强度增强,ΔΨm去极化,ROS及NF-κB的表达增高,细胞增殖增多、凋亡减少(P<0.05);而5-HD组与正常组相比,上述指标均无显著差异;(2)与正常组相比,Asthma组R-123荧光强度增强,ΔΨm去极化,ROS及NF-κB的表达增高,细胞增殖增多、凋亡减少(P<0.05);Asthma+diazoxide组与Asthma组相比,R-123荧光强度和ΔΨm去极化增强,ROS及NF-κB表达增高,细胞增殖增多、凋亡减少(P<0.05);Asthma+5-HD组与Asthma组相比,R-123荧光强度和ΔΨm去极化减弱,ROS及NF-κB表达降低,细胞增殖减少、凋亡增多(P<0.05);(3)各组大鼠ASMCs的R-123荧光强度,NF-κB mRNA表达量与ROS含量均呈正相关;MTT的吸光度值与NF-κB mRNA表达量呈正相关,凋亡细胞百分比与NF-κB mRNA表达量呈负相关。以上结果提示,哮喘可引起大鼠ASMCs的MitoKATP通道开放及ΔΨm去极化,ΔΨm去极化促进ROS的表达,ROS可能作为信号分子刺激NF-κB表达增加,进而促进ASMCs增殖,参与气道重构。更多还原

【Abstract】 The objective of this paper was to investigate the effect and mechanism of mitochondrial ATP-sensitive K+(MitoKATP) channel on the proliferation of airway smooth muscle cells(ASMCs) in asthmic rats.Thirty-six Sprague-Dawley(SD) rats were randomly assigned into 2 groups(18 in each):(1) Asthma group:the asthmic rat model was established by ovalbumin(OVA) sensiti-zation and excitation;(2) Normal group:rats were subjected to inhalation of equal amount of normal saline.The rat ASMCs were isolated from fresh lung tissues and cultured respectively as follows:(1) Control group:normal ASMCs were cultured under normoxia for 24 h;(2) Diazoxide group:normal ASMCs were cultured under normoxia for 24 h with diazoxide(an opener of MitoKATP channel);(3) 5-HD group:normal ASMCs were cultured under normoxia for 24 h with 5-hydroxydecanoate(5-HD)(an antagonist of MitoKATP channel);(4) Asthma group:Asthmic ASMCs were cultured under normoxia for 24 h;(5) Asthma + diazoxide group:Asthmic ASMCs were cultured under normoxia with diazoxide for 24 h;(6) Asthma + 5-HD group:Asthmic ASMCs were cultured under normoxia with 5-HD for 24 h.The mitochondrial membrane potential(ΔΨm) was detected using Rhodamine 123(R-123).The level of reactive oxygen species(ROS) was detected by DCF fluorescence.The expression of nuclear factor-kappa B(NF-κB) mRNA was examined by RT-PCR.The proliferation and apoptosis of rat ASMCs were examined respectively by MTT colorimetric assay and cell cycle analysis.The results were as follows.(1) After exposure to diazoxide for 24 h,the R-123 fluorescence intensity,the ROS level,NF-κB mRNA expression and the MTT absorbance value(A value) in normal ASMCs were significantly increased,and the apoptosis of rat ASMCs was significantly decreased compared to the control group(P<0.05).However,there was no significant changes in those indices after the normal ASMCs had been exposed to 5-HD for 24 h.(2) In Asthma and Asthma + diazoxide groups,the R-123 fluorescence intensity,ROS level and the MTT A value were markedly increased,and the apoptosis was markedly decreased compared to control group(P<0.05).These changes were more obvious in Asthma + diazoxide group than those in Asthma group(P<0.05).5-HD partly weakened the effect of asthma on the R-123 fluorescence intensity,ROS level and the MTT A value and the apoptosis of rat ASMCs(P<0.05).R-123 fluorescence intensity and NF-κB mRNA expression were positively correlated with ROS level.NF-κB mRNA expression was positively correlated with the MTT A value and negatively correlated with the apoptosis of rat ASMCs.All the results suggest that the opening of MitoKATP channel followed by a depolarization of ΔΨm contributes to the increase in ROS level and NF-κB mRNA expression in rat ASMCs and to the unbalance between cell proliferation and apoptosis of ASMCs induced by asthma.This might be a mechanism of the development of airway remodeling in asthma.更多还原