外泌体介导miRNA-29b在胆道闭锁肝纤维化中的作用与机制研究

Biliary atresia (BA) is a life-threatening cholestatic disease and its cause is still unknown. Bile duct damage and progressive liver fibrosis after operation is the main reason for the poor prognosis. The mechanism of liver fibrosis is also obscure. Epithelium-mesenchymal transition (EMT) has been found to play an important role in the pathogenesis mechanism of liver fibrosis with BA recently and many signaling pathways are activated. Exosomes involve in the pathogenesis of a variety of liver diseases. Our previous study found that there were a lot of exosomes that contained a larger number of miRNA-29b in peripheral blood in BA patients. DNA hypomethylation in the genome was found in liver with BA children. The expressions of DNA methylases (DNMTs) were obviously down-regulated. Epigenetics demonstrated that DNA methylation and DNMTs could be regulated by micro ribonucleic acid (microRNA, miRNA). MiRNA-29b can regulate DNMTs by targeting DNMTs mRNAs. Therefore, we assume that there exits an molecular mechanism in the pathogenesis of liver fibrosis with BA, that is “miRNA-29b overexpression in exosomes in peripheral blood – DNMTs downregulation – DNA hypomethylation in the promoter region of genes of EMT – EMT – liver fibrosis”. The following study is to verify the mechanism in there levels of human blood and tissue, cell and animal model. This study will be important for unclosing the mechanism of pathogenesis in liver fibrosis with BA.

胆道闭锁（biliary atresia, BA）是严重威胁儿童健康的胆汁淤积性疾病，胆管破坏和进行性的肝纤维化是预后差的主要原因。最近认为上皮间质转分化，可能是参与BA进行性肝硬化的一种病理机制，外泌体参与了肝脏纤维化的发病。本项目组前期研究发现，BA患儿外周血存在大量富含miRNA-29b的外泌体，肝脏基因组整体甲基化水平降低。表观遗传学研究发现DNA低甲基化和DNMTs受到微核糖核酸的调控。而miRNA-29b可靶向抑制DNMTs mRNA的翻译。因此我们推测，在BA肝纤维化的发病中存在“外周血外泌体miRNA-29b高表达—DNMTs抑制—上皮间质转分化相关基因启动子区低甲基化—上皮间质转化—肝纤维化”这一机制。本课题将从外周血、细胞和动物模型三个层次验证外泌体介导的miRNA抑制DNMTs促进上皮间质转分化，进而肝纤维化这一机制。本课题对揭示BA肝纤维化的发病机制具有重要意义。

胆道闭锁（biliary atresia, BA）是发生在婴幼儿时期阻塞性黄疸最常见的病因之一，也是婴幼儿最常见的肝脏疾病。其病理改变特征是进行性破坏性的肝胆管炎症和纤维性梗阻，肝内外胆道纤维化闭锁，最终发生肝硬化，是胆管破坏和进行性的肝纤维化是预后差的主要原因，严重威胁儿童健康。最近研究认为，外泌体参与了肝脏纤维化的发病。本项目组前期研究发现，BA患儿外周血存在大量富含miRNA-29b的外泌体，肝脏基因组整体甲基化水平降低。表观遗传学研究发现DNA低甲基化和DNMTs受到微核糖核酸的调控，而miRNA-29b可靶向抑制DNMTs mRNA的翻译。因此我们从人体外周血、细胞和动物模型三个层次研究，在BA肝纤维化的发病中存在“外周血外泌体miRNA-29b高表达—DNMTs抑制---相关基因启动子区低甲基化—上皮间质转化—肝纤维化”这一机制。通过收集胆道闭锁病人外周血，并提取外泌体，检测发现miRNA-29b高表达，进一步构建miR-29b过表达腺病毒载体转染jurkat细胞，提取转染前后细胞的外泌体，将提取的外泌体分别处理人正常肝细胞L-02和人肝内胆管上皮HIBEpic 细胞，检测发现miR-29b高表达，高表达miR-29b的外泌体可以抑制L-02细胞和HIBEpic 细胞中DNMT3a、DNMT3b的表达，促进Snail、Fibronectin、α-SMA的表达，促进cleaved caspase-3、Bax、p21的表达，抑制Ki67、PCNA的表达。同时抑制小鼠胆道闭锁模型肝组织中miR-29b的表达，可以改善小鼠的肝功能，减轻小鼠肝组织的纤维化情况，促进肝细胞的增殖和抑制肝细胞的凋亡。因此，得出结论，高表达的miR-29b的外泌体可以通过抑制L-02细胞和HIBEpic 细胞中DNMTs的表达，促进EMT相关基因的启动子低甲基化，促进EMT相关基因的表达，进而促进胆道梗阻型肝纤维化的发病进程。这一发现揭示BA肝纤维化的发病机制，对BA的发病及早期诊治的探索具有重要意义。