JNK对转化生长因子β1所致大鼠腹膜间皮细胞转分化的调控作用  

JNK regulates epithelial mesenchymal transition induced by transforming growth factor β1 in rat peritoneal mesothelial cells

作　　者：刘庆华[1] 余学清[1] 聂静[1] 毛海萍[1] 周飞宇[1] 李晓艳[1] 骆宁[1] 董秀清[1] 

机构地区：[1]中山大学附属第一医院肾内科,广州510080

出　　处：《中华肾脏病杂志》2008年第7期487-492,共6页Chinese Journal of Nephrology

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）诱导大鼠腹膜间皮细胞（RPMC）转分化中的调控作用。方法采用腹腔注射胰蛋白酶法分离培养RPMC，取第2代腹腔间皮细胞用于实验研究。观察TGF-β1对α平滑肌肌动蛋白（α—SMA）、Ⅰ型胶原（ColⅠ）、E钙黏蛋白（E—cadherin）以及磷酸化（p）JNK表达的影响；应用JNK特异性抑制剂SP600125预处理细胞后，观察其对TGF-β1致上述作用的影响。RT-PCR法检测α—SMA、ColⅠ及E-cadherinmRNA表达；Western印迹法检测α-SMA、ColⅠ、E—cadherin及p-JNK蛋白表达；间接免疫荧光检测α—SMA在细胞内的表达和分布。结果TGF-β1激RPMC能导致α-SMA、ColⅠ蛋白表达上调，E-cadherin蛋白表达下调，呈时间依赖性。TGF-β1刺激RPMC 5min出现p-JNK表达上调，10min达高峰（P〈0．01）。SP600125能够抑制JNK的磷酸化（P〈0．05），也能抑制TGF-β1导的α-SMA、ColⅠ mRNA和蛋白的高表达以及E-cadherin表达的下调（均P〈0．05）。间接免疫荧光结果显示，TGF－β1刺激RPMC48h，胞内α-SMA表达明显增多，SP600125能有效抑制其高表达。结论JNK在TGF-β1诱导大鼠腹膜间皮细胞转分化中具有重要的调控作用，JNK特异性抑制剂的应用可能为临床防治腹膜纤维化提供新的途径。Objective To investigate the role of C-Jun N-terminal kinase （JNK） in epithelial mesenchymal transition （EMT） induced by transforming growth factor β1 （TGF-β1） in rat peritoneal mesothelial cells（RPMCs）. Methods RPMCs were harvested from the peritoneum of male Sprague-Dawley rats, then cultured in DMEM/F12 medium with 15% （V/V） FBS. After stimulation with TGF-β1, the expression of a-smooth muscle actin （α-SMA）, E-cadherin and collagen I were detected in RPMCs. In some groups, the cells were pretreated with SP600125, a specific inhibitor of JNK, for 4 hours before incubation with TGF-β1. The protein expression of phosphorylated JNK was detected by Western blotting. The mRNA and protein expression of α-SMA, E-cadherin and collagen Ⅰ were examined with RT-PCR and Western blotting, respectively. The intracellular distribution and expression of α-SMA was determined by indirect immunofluorescence. Results TGF-β1 could significantly increase the expression of α-SMA and collagen Ⅰ, and decrease the expression of E-cadherin in RPMCs. TGF-β1 could stimulate the expression of phosphorylated JNK at 5 minutes with the peak at 10 minutes （P〈0.01）. The addition of SP600125 effectively inhibited TGF-β1-induced high expression of α-SMA and collagen Ⅰ （P〈 0.05）, and prevented TGF-β1-induced down-regulation of E-cadherin expression in RPMCs （P〈 0.05）. The indirect immunofluorescence showed that the expression of intracellular α-SMA in RPMCs stimulated by TGF-β1 for 48 h increased significantly, which could be inhibited by SP600125. Conclusions JNK regulates epithelial mesenchymal transition induced by TGF-β1 in rat peritoneal mesothelial cells. JNK inhibitor may be used as a novel therapeutic agent for peritoneal fibrosis.

关 键 词：转化生长因子β 纤维化 JNK丝裂原活化蛋白激酶类 上皮间质转分化 间皮细胞 

分 类 号：R285.5[医药卫生—中药学]