容量激活性氯离子通道在大鼠低氧性PASMCs中的表达及与p38MAPK通路的关系

Expression of Volume Activated Chloride Channel in Hypoxic Pulmonary Artery Smooth Muscle Cell of Rat and Its Relationship with p38MAPK Signal Pathway

目的探讨容量激活性氯离子通道(CLC3)在大鼠低氧性肺动脉平滑肌细胞(PASMCs)中mRNA和蛋白表达的变化以及其与p38MAPK信号通路的关系。方法取10只雄性SD大鼠,用酶消化法获得PASMCs并且进行原代培养,用小鼠抗大鼠SMα-actin免疫荧光细胞化学的方法进行鉴定。经培养鉴定的PASMCs随机分为:常氧组(N组,n=6),低氧组(H组,n=6),DMSO对照组(D组,n=6),SB203580干预组(SB组,n=6),Anisomycin干预组(A组,n=6),用免疫印迹技术检测CLC3蛋白的表达,半定量反转录-聚合酶链反应(RT-PCR)技术测定CLC3 mRNA水平的表达。结果与N组比较,H组PASMCs中CLC3 mRNA和蛋白的表达量均显著上调(P均<0.01);与D组比较,SB组CLC3 mRNA和蛋白的表达均上调(P均<0.01),A组mRNA的表达明显下调(P<0.01),蛋白的表达轻微下调(P>0.05)。结论低氧可上调大鼠PASMCs中CLC3 mRNA和蛋白的表达;p38MAPK通路抑制剂SB203580能上调PASMCs中CLC3 mRNA和蛋白的表达;p38MAPK通路激活剂Anisomycin可下调PASMCs中CLC3 mRNA和蛋白的表达。

Objective To investigate the expression of volume activated chloride channel （CLC3） in hypoxic pulmonary artery smooth muscle cell （PASMCs） of rat and its relation with p38MAPK signal pathway.Methods PASMCs were obtained from ten male SD rats by the enzyme digestion method,and the following generations were cultured in serum contained DMEM medium.The second to sixth generation cells were confiemed by immunofluorescence cytochemistry method.And these PASMCs were randomly divided into 5 groups including normal group（N group,n =6）,hypoxia group（H group,n =6）,DMSO group（D group,n =6）,SB203580 group （SB group,n =6） and Anisomycin group（A group,n =6）.The expression of CLC3 in PASMCs was detected by Western blotting method.The mRNA expression of CLC3 was detected by half quantitative reverse transcription polymerase chain reaction （RT-PCR）.Results The mRNA and protein expressions of CLC3 in H group were significantly higher than N group （P ＜0.01）.Compared with D group,the mRNA and protein expressions of CLC3 were significantly increased in SB group （P ＜ 0.01）,and the mRNA expression of CLC3 in A group was obviously increased,（P ＜ 0.01） with slightly decreasing of its protein expression （P ＞ 0.05）.Conclusion Hypoxia promotes the expressions of mRNA and protein of CLC3 in rat PASMCs.The p38MAPK pathway inhibitor SB203580 induces the upregulation of the mRNA and protein expressiosn of CLC3 in rats PASMCs.And.p38MAPK pathway activator Anisomycin reduces the mRNA and protein expression of CLC3 in rats PASMCs.