表达持续活化型ALK3抑制大鼠肝星状细胞活化

Stable expression of constitutively activated ALK3 suppresses rat hepatic stellate cell activation

背景肝纤维化与肝星状细胞(hepatic stellate cells, HSCs)上皮细胞间充质转化(epithelial mesenchymal transformation,EMT)相关, TGF-β1在其中起着重要的作用,而BMP7能够拮抗TGF-β1,目前主要研究的是TGF-β/BMPs-Smad信号通路, ALK3属于BMPs的持续活化Ⅰ型受体,在肝纤维化分子研究机制中较少运用.目的观察大鼠HSCs中稳定表达持续活化型ALK3时Sma d1磷酸化及纤维化相关基因E-cadherin、α-SMA、col1A2等表达情况,探讨BMP-7信号转导在肝纤维化发生发展过程中拮抗TGF-β1信号及其抗肝纤维化机制.方法建立体外培养大鼠HSCs (HSC-T6)持续活化型ALK3的稳定表达细胞株, MTT检测细胞的增殖;RT-PCR检测col1A2等相关分子mRNA水平;Western blotting检测Samd1、E-cadherin、α-SMA、col1A2蛋白表达和Smad1磷酸化;显微镜下观察细胞形态.结果稳定表达持续活化型ALK3的HSC-T6细胞增殖受到抑制, Smad1磷酸化显著升高,α-SMA, col1A2表达下调, E-cadherin表达上调.结论BMP-7信号转导通过增强Samd1的磷酸化而拮抗TGF-β1致纤维化作用,抑制大鼠HSCs的活化.

BACKGROUND Hepatic fibrosis is related to activation of hepatic stellate cells (HSCs) and epithelial mesenchymal transformation (EMT), in which transforming growth factor-β1 (TGF-β1) plays a pivotal role, but bone morphogenetic protein-7 (BMP7) can antagonize TGF-β1. Currently, the TGF-β/BMPs- Smad signaling pathway is a hot topic of research in this field. ALK3 belongs to the constitutively activated type Ⅰ receptor of BMPs, and its role in the molecular mechanism of hepatic fibrosis is rarely studied. AIM To detect the expression of Samd1, P-Smad1, and fibrosis-related genes E-cadherin,α-SMA, and col1A2 in cultured rat HSCs (HSC-T6) to investigate how BMP-7 antagonizes TGF-β1 in the development of liver fibrosis and its anti-hepatic fibrosis mechanisms. METHODS After HSCs-T6 were transfected with constitutively active cDNA construct expressing ALK3, RTPCR method was used to screen the cell line with stable ALK3 expression and detect the mRNA level of col1A2. MTT assay was used to examine the proliferation of HSC-T6 cells with high expression of ALK3. Western blot method was used to detect the expression of Smad1, P-Smad1, E-cadherin,α-SMA, and co1lA2. Optic microscopy was used to detect the morphological changes of HSC-T6 cells with high expression of ALK3. RESULTS Compared with control cells, ALK3 high expression restrained the growth of HSC-T6 cells, suppressed the expression of α-SMA and col1A2, promoted the expression of P-Smad1 and E-cadherin, but had no significant effect on Samd1. CONCLUSION BMP-7 competitively antagonizes TGF-β1 induced fibrosis by enhancing the phosphorylation of Samd1.