胆道闭锁中miR-21介导PTEN信号通路对肝纤维化的作用机制

Biliary atresia(BA),an inflammatory sclerosing chllangiopathy, is the leading severe liver disease in infancy. It is featured by serious portal fibrosis. Although Kasai procedure establishes biliary drainage, liver fibrosis still processes until liver transplantation on the long term follow-up. We observed evaluated miR-21 expression in previous microRNA chips work, and it is verified with increasing miR-21 and decreasing target gene PTEN through PCR. miR-21's expression mainly located in the portal area from literature. We injected the BA murine model with antagomir intraperitoneally, and the portal fibrosis was relieved. Myofibroblast's activation and biliary epithelial-mesenchymal transition may contribute in the portal fibrosis, and miR-21's regulation has not been reported. We intend to culture the myofibroblasts and biliary epithelial cells in vitro, and detect miR-21、PTEN and downstream molecule's expression. Treating by antagomir and LY294002 at different link，we plan to investigate the regulation between the pathway and portal fibrosis. After all we will assess the effect of antagomir and ly294002 on liver fibrosis in BA animal model. We expect to improve patients' progonosis through this research.

胆道闭锁（BA）以肝内外胆管进行性炎症和纤维性梗阻为特征，汇管区纤维化非常明显。术后肝纤维化持续进展，往往需要接受肝移植。miRNA芯片中发现BA肝组织中miR-21表达升高，PCR验证了miR-21的升高和靶基因PTEN的下降。文献报道miR-21高表达于汇管区周围。我们对BA模型小鼠腹腔内注射miR-21拮抗剂antagomir后，观察到汇管区的纤维化程度减轻。汇管区纤维化的机制包括:肌纤维母细胞的活化和胆管上皮间充质转化，目前miR-21对两者的调控均无文献报道。本课题拟进一步体外培养BA患儿的肌纤维母细胞及胆管上皮，观察miR-21介导PTEN信号通路在其中的表达。在不同环节分别给予antagomir、LY294002处理，验证miR-21对肌纤维母细胞活化和胆管上皮间质转化的调控作用。在初步明确机制后，在动物模型中验证作用机制及评估治疗作用。

背景：微小RNA是一组在转录后水平对靶基因进行负调控的短链、非编码RNA分子。越来越多的研究表明微小RNA参与了肝脏纤维化进程。胆道闭锁肝脏纤维化有着持续迅速进展的特点。本研究着重于微小RNA-21在胆道闭锁病理过程中的作用。.方法：我们分别收集胆道闭锁及肝脏外伤的肝脏标本作为实验组和对照组。我们用实时荧光定量PCR方法检测微小RNA-21、PTEN及a-SMA在肝脏组织中的RNA表达量。通过Western blot和免疫组化的方法分析PTEN、a-SMA及磷酸化AKT在肝脏中的蛋白表达含量。.结果：我们发现在胆道闭锁病人肝脏中微小RNA-21表达上调，通过3‘非编码区负性调控PTEN后，活化了下游的AKT通路促进了肝脏纤维化，a-SMA表达升高。.结论：微小RNA-21/PTEN/AKT轴心在胆道闭锁肝脏纤维化进程中发挥了促进作用，可以作为一个潜在的治疗靶点。