长链非编码RNA-BC090353调控IL6表达对胆道闭锁肝内胆管增生机制研究

Abnormal hyperplasia of intrahepatic bile duct is an important factor for the rapid progress of liver fibrosis in biliary atresia (BA). Presently, prevention and treatment of biliary hyperplasia is still very difficult because of the unclear pathogenesis. Our previous studies have suggested that the long non-coding RNA-BC090353 and IL6 play an important role in the hyperplasia of intrahepatic bile duct of BA patients, but the mechanisms involved are unclear. Bioinformatics predicts that BC090353 can regulate the expression of IL6 by disrupting the NF-kB-mediated IL6 transcription and sponge miR-200s. We propose that BC090353 regulates the expression of IL6 in the form of transcription factors and molecular sponges, which further influence the intrahepatic bile duct hyperplasia of BA. To test this hypothesis, we will analyze the roles of BC090353 in the intrahepatic bile duct hyperplasia of BA on the levels of molecules, cells, tissues and even whole animal; We next to explore the regulatory mechanism of BC090353 expression; We further to determine the effect of BC090353 on intrahepatic bile duct hyperplasia related genes; Finally, we will reveal the molecular mechanism of BC090353 in regulating the expression of IL6. This study will explore the pathogenesis of the abnormal hyperplasia of bile duct in the liver from BA. Our study will be helpful in revealing the mechanism of for BA liver fibrosis and provide new ideas for clinical prevention and treatment from a new perspective BC090353.

肝内胆管异常增生是造成胆道闭锁（BA）肝纤维化进展迅速的重要原因，其发生机制不明，临床干预困难。我们前期研究结果提示肝脏长链非编码RNA-BC090353 及IL6在BA肝内胆管增生中起重要作用，但作用机制不清楚。生物信息学预测BC090353能通过竞争结合NF-kB及海绵吸附miR-200s调节IL6转录水平。据此我们提出假说：BC090353以转录调控因子和分子海绵的形式共同调控IL6表达，参与BA肝内胆管增生。为了验证这一假说，本课题拟从分子、细胞、组织和动物整体水平等多层面明确BC090353在BA肝内胆管增生中的具体作用；探讨BC090353表达的调控机制；确定BC090353对肝内胆管增生相关基因的调控；揭示BC090353调控IL6表达的分子机制。本研究将从BC090353这个新视点探讨BA肝内胆管异常增生的发生机制，为BA肝脏纤维化机制奠定基础，为临床防治提供新思路。

胆道闭锁(Biliary Atresia, BA)是发生于婴幼儿时期的一种胆汁淤积疾病，是造成婴幼儿死亡的主要肝脏疾病。BA的一个重要病理学特征是肝纤维化进展迅速，明显超过成人和婴幼儿其他疾病，如果无有效干预治疗，肝硬化会在出生后几周内出现。肝内胆管异常增生是造成BA肝纤维化进展迅速的重要原因，其发生机制不明，临床干预困难。根据前期研究结果提示，我们首先发现了BDL处理后，大鼠肝内胆管大量增生且伴随肝脏长链非编码 RNA（long non-coding RNA, lncRNA ）表达谱、microRNA（miRNA）表达谱及基因表达谱发生了巨大变化：BDL大鼠肝内长链非编码RNA-BC090353表达降低，IL6基因表达升高而Foxa2基因表达下降，miR-200家族表达升高。为了研究lncRNA-BC090353在胆汁淤积肝损害中的具体作用。我们紧接着进行了依据研究计划构建了BC090353腺相关病毒血清9型（AAV9）过表达腺病毒。定向过表达BC090353于大鼠肝脏中，发现在BDL大鼠模型中，通过AAV9过表达BC090353加重肝损伤程度。研究结果进一步发现BC090353 及IL6表达水平在BDL诱导下显著升高。BDL诱导胆管增生及肝纤维化过程中IL6/STAT3信号通路显著活化。大鼠体内过表达BC090353加速了BDL大鼠胆管增生及肝纤维化进程，且进一步激活IL6/STAT3信号。我们还进行了深化研究。我们发现胆道闭锁（BA）患儿肝脏炎性巨噬细胞数量在肝胆损伤部位募集和发生炎症反应如产生IL6等炎症因子。条件性敲除巨噬细胞能够显著地改善胆汁淤积性肝损害和肝纤维化进程。另外，我们还检测了患儿肝脏中其他lncRNAs的表达变化。结果发现lncRNA-H19、lncRNA-00029、lncRNA-00312及lncRNA-00526在BA患儿肝内显著性升高。体内敲除lncRNA-H19减轻了胆汁淤积性肝损害。综合以上所述，我们研究揭示了BC090353调控IL6/STAT3信号的分子机制。本研究将从BC090353这个新视点探讨BA肝内胆管异常增生的发生机制，为BA肝脏纤维化机制奠定基础，为临床防治提供新思路。