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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 β1的促表型分化作用
Objective To investigate the role of mitogen-activated protein kinase (MAPK) in the phenotypic differentiation of human lung fibroblasts induced by transforming growth factor β1 (TGFβ1). Methods Human lung fibroblasts HLF 0 2 cell line was treated with 10 ng / ml TGF β1 for different times. Blocking experiments blocked the p38 pathway and the expression of extracellular regulated kinase (Erk) by SB2 0 35 80 and PD980 5 9 respectively. The expression of α-smooth muscle actin (αSMA) mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of p38, Erk, and jun amino-terminal kinase (JNK) phosphorylation and the intensity of alphaSMA protein expression. Results (1) The expressions of αSMA mRNA in HLF 0 2 cells treated with TGF β1 for 24 h, 48 h and 72 h were 1.87 ± 0.11, 2.49 ± 0.10, 3.02 ± 0.15, αSMA protein expression levels were: 3.2 0 ± 0.14, 3.96 ± 0. 2 1,4. 57 ± 0.13. (2) TGF-β1 can activate the p38 and Erk pathway in HLF 0 2 cells without activation of JNK. (3) SB2 0 35 80, PD980 5 9 each inhibited TGF β1 -induced phosphorylation of p38, Erk kinase. (4) SB203580 inhibited the expression ofαSMA mRNA and protein induced by TGFβ1 (30% and 40% inhibition respectively); PD98059 also inhibited the expression ofαSMA mRNA and protein induced by TGFβ1 (the inhibitory rates were 10 % And 20%). Conclusion TGF β1 induces phenotypic differentiation in HLF 0 2 cells. P38 and Erk kinase are involved in the regulation of TGF β1 phenotypic differentiation