TNFα诱导蛋白3在人真皮成纤维细胞向肌成纤维细胞表型转化中的作用及机制

摘要

目的 探究TNFα诱导蛋白3(A20蛋白)在人真皮成纤维细胞(Fb)向肌成纤维细胞(MFb)表型转化中的作用及机制.方法 通过酶消化法分离培养原代人正常皮肤Fb (NFb)和瘢痕皮肤Fb(HFb),取对数生长期3～5代细胞进行试验,转化生长因子-β(TGF-β)刺激Fb模拟Fb向MFb转化过程,采用细胞转染敲除Fb中A20的表达,qRT-PCR、Western blot检测Fb中A20、Ⅰ型胶原(Col Ⅰ)、Ⅲ型胶原(Col Ⅲ)和α-平滑肌肌动蛋白(α-SMA)、p-Smad 2等变化.结果 与NFb相比,HFb中A20低表达(P ＜0.05);TGF-β刺激Fb后,A20表达显著降低(P＜0.05),Col Ⅰ、Col Ⅲ及α-SMA等纤维化相关分子表达显著升高(P＜0.05),p-Smad 2表达显著升高(P＜0.05);敲除Fb中的A20,可进一步促进TGF-β诱导的Col Ⅰ、Col Ⅲ及α-SMA等纤维化相关分子及p-Smad 2表达的升高(P＜0.05).结论 A20蛋白参与了人真皮Fb向MFb表型转化的过程,并通过抑制Smad信号通路的活化,对TGF-β所介导的转化效应起负性调控作用.%Objective To investigate the role and mechanism of A20 protein in the phenotypic transformation of human dermal fibroblasts (Fb) to myofibroblasts (MFb).Methods Primary human normal skin Fb (NFb) and scar skin Fb (HFb) were isolated and cuhured by enzymatic digestion.The cells were used in logarithmic growth phase at 3-5 passages,and Fb was stimulated by transforming growth factor-β (TGF-β) to mimic the Fb to MFb transformation process.Cell transfection was conducted to knock out the expression of A20 in Fb,and quantitative real-time polymerase chain reaction (PCR) test,protein immunoblotting test were conducted to detect A20,type Ⅰ collagen (Col Ⅰ),type Ⅲ collagen (Col Ⅲ)and α-smooth muscle (α-SMA),and p-Smad 2 expression in Fb.Results Compared with normal skin Fb,A20 was down-regulated in pathological scar Fb (P ＜ 0.05).After TGF-β stimulated,A20 expression was significantly decreased (P ＜ 0.05),while the expression of fibrosis associated molecules such as Col Ⅰ,Col Ⅲ and α-SMA and p-Smad 2 was significantly increased (P ＜ 0.05) in Fb.Knockout of A20 in Fb further promoted TGF-β induced the expression of fibrosis associated molecules such as Col Ⅰ,Col Ⅲ and o-SMA and p-Smad 2 (P ＜ 0.05).Conclusions A20 protein is involved in the process of human dermal Fb to MFb phenotype transformation,and A20 negatively regulates the transformation effect mediated by TGF-β by inhibiting the activation of Smad signaling pathway.