转化生长因子β1诱导皮肤成纤维细胞向肌成纤维细胞表型转分化的机制研究

TGF-β1-induced transdifferentiation of derma fibroblasts into myofibroblasts:a study of mechanism

目的:探讨TGF-β1诱导皮肤成纤维细胞(FB)向肌成纤维细胞转分化的可能途径和调控机制。方法:将野生型和Smad3基因敲除(KO)型小鼠皮肤FB分为9组:野生型FB组、野生型FB+TGF-β1组、野生型FB+SB431542组、野生型FB+SB431542+TGF-β1组、Smad3KOFB组、Smad3KOFB+TGF-β1组、野生型FB+SB203580+TGF-β1组、野生型FB+PD98059+TGF-β1组和野生型FB+SP600125+TGF-β1组。各组细胞经同步化处理后,直接以TGF-β1刺激或经上述各激酶抑制剂预处理后再以TGF-β1刺激。收集细胞,一部分以单细胞RT-PCR检测α-SMA阳性表达百分比,另一部分细胞抽提总RNA后采用实时荧光定量RT-PCR检测α-SMA的表达水平变化。结果:Smad3KO组与SB431542组的α-SMA表达水平和阳性百分比显著升高(Smad3KOFB组vs野生型FB组;野生型FB+SB431542+TGF-β1组vs野生型FB+SB431542组;Smad3KOFB+TGF-β1组vsSmad3KOFB组,P<0.01),而SB203580组和SP600125组中α-SMA表达水平和阳性百分比升高的作用则被显著抑制(野生型FB+SB203580+TGF-β1组、野生型FB+SP600125+TGF-β1组vs野生型FB+TGF-β1组,P<0.05)。结论:在TGF-β1诱导成纤维细胞向肌成纤维细胞的转分化过程中,Smad3途径介导抑制作用,而p38/MAPK、JNK/MAPK途径则介导正向调节作用。

Objective：To explore the possible pathways and regulatory mechanism of TGF-β1-induced transdifferentiation of derma fibroblasts（FB） into myofibroblasts. Methods.. Mice Wild-type and Smad3 knockout（Smad3 KO） derma FB were divided into 9 groups,namely,A： Wild-type FB; B： Wild-type FB＋TGF-β1 ; C ： Wild-type FB＋ SB431542 ; D： Wild-type FB＋ SB431542 ＋ TGF-β1 ; E ： Smad3 KO FB; F ： Smad3 KO FB＋ TGF-β1 ; G ： Wild-type FB＋ SB203580 ＋ TGF-β1 ; H ： Wild-type FB＋ PD98059 ＋TGF-β1; and Ⅰ. Wild-type FB＋ SP600125 ＋ TGF-β1. After synchronization treatment, the cells were treated with TGF-β1 with or without pretreatment with above mentioned kinases inhibitors. Then the cells were collected for RNA extraction and the expression of α-SMA was detected by real time quantitative RT-PCR; some cells were analyzed by single cell RT-PCR to test the positive expression rate of α-SMA. Results.. The expression and positive rate of α-SMA in SB431542 group and Smad3 knockout group were significantly increased（group E vs. group A; group D vs. group C; group F vs. group E, P〈0. 01 ） and those in SP600125 group and SB203580 group were significantly inhibited（group G and I vs. group B,P〈0.05）. Conclusion： In TGF- β1-induced derma fibroblasts transdifferentiation into myofibroblasts,Smad3 pathway plays a negative regulatory role and p38/ MAPK and JNK/MAPK pathway play a positive regulatory role.