JNK对TGF-β1诱导的人肺上皮-间质转分化的调控作用

摘要

目的：探讨c‐Jun氨基末端激酶（JNK）在转化生长因子‐β1（TGF‐β1）诱导的人肺上皮细胞A549转分化中的调控作用。方法将体外培养的人肺上皮细胞（A549）随机分成3组：正常对照组、TGF‐β1组（加入10 ng／mL TGF‐β1）及抑制剂组（加入10 ng／mL TGF‐β1和20μmol／L JNK的特异性抑制剂Sp600125），培养于3％的血清培养液中，光镜下观察3组细胞形态的变化，并通过RT‐PCT检测各组A549细胞的上皮标志物E‐钙黏蛋白（E‐cadherin ，E‐cad）及间充质标志物α‐平滑肌肌动蛋白（α‐smooth muscle actin ，α‐SMA）和胶原纤维Ⅰ（collagen fibersⅠ，ColⅠ）的表达的变化，West‐ern blot检测JNK磷酸化（p‐JNK）水平的变化。结果正常对照组体外培养的A549细胞光镜下为鹅卵石样紧密排列生长，有E‐cad表达及微量的α‐SMA、ColⅠ及p‐JNK表达。TGF‐β1组培养72 h后细胞基本长成梭形、纺锤形，E‐cad表达下调，α‐SMA、ColⅠ及p‐JNK表达上调。与 TGF‐β1组比较，抑制剂组培养72 h后细胞梭形有所逆转，E‐cad表达上调，α‐SM A、ColⅠ及p‐JNK表达明显抑制；与正常对照组比较，细胞形态较扁长，E‐cad、α‐SM A、ColⅠ及p‐JNK表达差异无统计学意义。结论 JNK信号通路参与 TGF‐β1介导的人肺上皮‐间质转分化过程，JNK的特异性抑制剂Sp600125可有效抑制该过程。%Objective To explore the role of JNK signaling pathway in epithelial mesenchymal transition (EMT)process of human alveolar epithelial cells A549 induced by TGF‐β1 in vitro.Methods Human alveolar epithelial cells (A549)cultured in vitro were divided randomly into three groups :normal group ,TGF‐β1 group ( treated by TGF‐β1 with 10 ng/mL)and inhibitor group (treated by 10 ng/mL TGF‐β1 and 20 μmol/L Sp600125).Morphological observation on the cells was performed under light microscope after culturing in 3% serum medium for 72 h. The expression of E‐cadherin (E‐cad ,a epithelial cell marker) ,α‐smooth muscle actin (α‐SMA ,a mesenchymal cell marker)and Collagen fibers Ⅰ(ColⅠ ,a mesenchymal cell marker)were tested by RT‐PCR.The level of JNK phosphorylation (p‐JNK)was detected through Western blot.All experiments were repeated three times at least.Results The normal human alveolar epithelial cells (A549)cultured invitro were arranged closely like peb‐bles.E‐cad expressed at a certain level ,while the expression ofα‐SMA ,ColⅠand p‐JNK was weakly detected.In TGF‐β1 group , the cells were spindle‐shaped ,the expression of E‐cad was reduced ,while the expression ofα‐SMA ,ColⅠand p‐JNK was signifi‐cantly increased 72 h after treatment of TGF‐β1. However ,compared with TGF‐β1 group ,spindle‐shaped cells in the inhibitor group were recovered after 72 h being treated by TGF‐β1 and Sp600125 ,the expression of E‐cad was increased and the expres‐sion levels of α‐SMA ,ColⅠand p‐JNK were significantly decreased 72 h after treatment with TGF‐β1 and Sp600125 in inhibitor group. As compared with normal group ,the shape of the cells in inhibitor group was prolate ,and the expression of E‐cad ,α‐SMA ,ColⅠand p‐JNK was not significantly different.Conclusion JNK signaling pathway is related to the process of EMT of human alveolar epithelial cells induced by TGF‐β1. Sp600125 ,a special inhibitor of JNK ,could validly restrain the process.