BMP-9在胆道闭锁肝纤维化中的作用机制研究

目的 探讨骨形态发生蛋白9（BMP-9）在胆道闭锁（BA）肝纤维化中的作用机制。方法 选取14例BA患 儿肝组织标本为BA组，5例胆总管囊肿（CBD）患儿肝组织标本为CBD组，HE染色对肝组织进行肝纤维化评估，免疫 组化染色检测BMP-9、p-SMAD1/5表达情况，实时荧光定量逆转录聚合酶链反应（qPCR）检测肝组织BMP-9及DNA 结合抑制因子 1（ID1）mRNA 表达水平。培养人肝星状细胞 LX-2，用重组转化生长因子（rTGF）-β1 与重组 BMP （rBMP）-9处理，蛋白质印迹法检测细胞中SMAD1/5、p-SMAD1/5、ID1蛋白及α-平滑肌肌动蛋白（α-SMA）表达情况， qPCR检测细胞中α-SMA及ID1 mRNA表达情况。结果 BA组肝组织中BMP-9及p-SMAD1/5蛋白、BMP-9及ID1 mRNA表达水平均高于CBD组；在BA组中重度肝纤维化患儿BMP-9蛋白、BMP-9及ID1 mRNA的表达高于轻度肝 纤维化患儿（P＜0.05）。LX-2细胞经rTGF-β1、rBMP-9处理后，α-SMA蛋白及α-SMA mRNA表达水平均升高（P＜ 0.05）。LX-2细胞加入不同剂量rBMP-9之后，其下游通路中p-SMAD1/5、SMAD1/5、ID1、α-SMA蛋白，及ID1、α-SMA mRNA表达均较0 μg/L组升高（P＜0.05）。结论 在BA患儿肝组织中BMP-9、p-SMAD1/5、ID1表达水平较CBD患 儿增高，与肝纤维化严重程度有关。BMP-9可通过SMAD/ID1信号通路激活人肝星状细胞LX-2，促进纤维化进程。

The study on the mechanism of BMP-9 in liver fibrosis of biliary atresia

Objective To explore the mechanism of BMP-9 in biliary atresia (BA) liver fibrosis. Methods Fourteen samples of BA cases were selected as the BA group and 5 samples of congenital biliary dilatation (CBD) cases were selected as the CBD group. Liver fibrosis was evaluated by HE staining, immunohistochemical staining was used to detect the expression levels of BMP-9 and p-SMAD1/5, and qPCR was used to detect the expression levels of BMP-9 and ID1 mRNA in liver. Human hepatic stellate cells (LX-2) were cultured and treated with rTGF-β1 and different concentrations of rBMP-9. The protein expression levels of α-SMA, SMAD1/5, p-SMAD1/5 and ID1 in cells were detected by Western blot assay, and the mRNA expressions of α-SMA and ID1 in cells were detected by qPCR. Results The expression levels of BMP-9 and p-SMAD1/5 proteins, BMP-9 and ID1 mRNA in liver were higher in the BA group than those of the CBD group. In the BA group, the expressions levels of BMP-9 protein, BMP-9 and ID1 mRNA were significantly higher in children with moderate and severe liver fibrosis than those in children with mild liver fibrosis (P＜0.05). After treatment with rTGF-β1 and rBMP-9, the expression levels of α-SMA protein and α-SMA mRNA were increased in LX-2 cells (P＜0.05). The expressions of p-SMAD1/5, SMAD1/5, ID1, α -SMA protein and ID1, α -SMA mRNA in the downstream pathway were increased after adding different concentrations of rBMP-9 in LX-2 cells compared with those of 0 μg/L group (P＜0.05). Conclusion The expression levels of BMP-9, p-SMAD1/5 and ID1 in the liver of BA children are increased than those of CBD children, which are positively correlated with the degree of liver fibrosis. BMP-9 can activate LX-2 cells through SMAD/ID1 signaling pathway and promote the progress of fibrosis.