转化生长因子β1和丝裂原活化激酶通路调控人肺成纤维细胞表型

Transforming growth factor-β1 induced phenotypic differentiation of human lung fibroblasts through mitogen activated protein kinase-dependent pathway

目的 探讨丝裂原活化蛋白激酶(MAPK)在转化生长因子β1(TGF β1)诱导人肺成纤维细胞表型分化中的作用。方法 以人肺成纤维细胞HLF 0 2细胞系为研究对象,给予10ng/mlTGF β1刺激不同时间;阻断实验以SB2 0 35 80、PD980 5 9分别阻断p38通路和细胞外调控激酶(Erk)通路;采用逆转录-聚合酶链反应(RT PCR)检测细胞内表型分化标志物α-平滑肌肌动蛋白(αSMA)mRNA的表达;运用Western印迹检测细胞内p38、Erk、c jun氨基末端激酶(JNK)磷酸化以及αSMA蛋白表达的强度。结果 (1)TGF β1刺激HLF 0 2细胞2 4h组、4 8h组、72h组的αSMAmRNA表达水平分别为:1.87±0 .11、2 .4 9±0 .10、3.0 2±0 .15 ;αSMA蛋白表达水平分别为:3.2 0±0 .14、3.96±0 .2 1、4 .5 7±0 .13。(2 )TGF β1可以激活HLF 0 2细胞内p38和Erk通路,对JNK没有活化作用。(3)SB2 0 35 80、PD980 5 9各自抑制了TGF β1诱导的p38、Erk激酶的磷酸化。(4)SB2 0 35 80抑制了TGF β1诱导的αSMAmRNA和蛋白表达(抑制率分别为30 %和4 0 % ) ;PD980 5 9同样抑制了TGF β1诱导的αSMAmRNA和蛋白表达(抑制率分别为10 %和2 0 % )。结论 TGF β1可诱导HLF 0 2细胞表型的分化,p38、Erk激酶参与了调控TGF

Objective To investigate the role of MAPK signal transduction in TGF-β1 induced phenotypic differentiation of human lung fibroblasts. Method Human lung fibroblasts cell line(HLF-02) were cultured and then stimulated with 10 ng/ml TGF-β1 for different time;SB203580 or PD98059 was added into culture medium to prevent p38 or Erk kinase pathway before incubating with TGF-β1 ;the expression of α-smooth muscle actin(α-SMA) was detected by Western blotting and RT-PCR;Western blotting was used to assay phosphorylation of p38,Erk,and JNK kinase. Results (1)In the process of stimulation by TGF-β1 ,the α-SMA mRNA expression levels of 24,48 and 72 h groups were 1.87±0.11,2.49±0.10,3.02±0.15 respectively;and the α-SMA protein expression levels of 24,48 and 72 h groups were 3.20±0.14,3.96±0.21,4.57±0.13 respectively.(2) TGF-β1 induced p38,Erk kinase phosphorylation but not JNK kinase.(3)The inhibitors SB203580 and PD98059 suppressed TGF-β1 -induced p38 kinase and Erk phosphorylation respectively.(4)SB203580 significantly attenuated TGF-β1 -induced α-SMA mRNA and protein expression(inhibition rate:30% and 40%);PD98059 also significantly inhibited TGF-β1-induced α-SMA mRNA and protein expression(inhibition rate:10% and 20%). Conclusion TGF-β1 is capable of inducing the phenotypic differentiation of HLF-02,which is regulated by p38 and Erk kinase signal pathway.