核转录因子Gli在胆道闭锁肝脏上皮间充质化中的调控机制研究

Biliary atresia is the main cause of the obstructive jaundice.So far, the pathogenesis is not clear.Early diagnosis of biliary atresia is very difficult, and it has become the primary cause of infant liver function failure in China. As a virus induced autoimmune disease, biliary atresia will finally lead to hepatic fibrosis and even death. Studies showed that epithelial mesenchymal transition (EMT) play a main role in liver fibrosis of biliary atresia. The cholangiocyte can convert into myofibroblast-like cells leading to liver fibrosis. It has been confirmed that the some signaling pathway could promote or reverse the hepatic EMT. Studies also demonstrated that Shh signaling pathway has a role in both biliary development and hepatic fibrosis.Transcriptional factor Gli is a terminal effector molecule of Shh signal pathway, and it directly regulate the target gene transcription and expression. As one of the target genes of transcriptional factor Gli, Snail is also a key regulatory factor in EMT process. So the transcriptional Gli is a potential target for studying the pathogenesis of biliary atresia. This research is intend to detect the gene expressions of Gli1/Gli2, Snail/Slug, and EMT cytokines of hepatic tissue in the patients and mice of biliary atresia, and to discover their relationships. With using the RNA interference technology and et al we will study the important regulatory role in the onset of the biliary atresia and the process of the hepatic EMT. In the end, the ultimate goal is to provide a new direction and target for biliary atresia prevention and treatment.

胆道闭锁是新生儿阻塞性黄疸的主要原因，发病机制不清，早期诊断率低,已成为我国婴幼儿肝功衰竭的首要致因。胆道闭锁是一类病毒诱导的自身免疫性疾病，最终导致肝纤维化而死亡。研究发现上皮间充质化（EMT）在胆道闭锁肝纤维化中起主导作用。胆管上皮细胞等可通过EMT转化为肌成纤维样细胞致纤维化发生，而通过相关信号通路的调控可促使或逆转EMT的发生。研究表明Shh信号在胆道发育和胆道闭锁肝EMT中均有明确的作用，作为Shh信号的终端效应分子，核转录因子Gli直接调控靶基因的转录表达。Snail是其靶基因之一，也是EMT的关键调控因子，故Gli转录因子是研究胆道闭锁发病的理想靶点。本课题拟通过检测胆道闭锁患儿及模型小鼠肝脏Gli、Snail基因及EMT特征因子的表达，明确其与胆道闭锁发病的相关关系后，应用RNAi等技术研究Gli在胆道闭锁发病及肝EMT中的重要调控作用，为胆道闭锁的防治提供新的思路和靶点。

研究背景：胆道闭锁是新生儿阻塞性黄疸的主要原因，发病机制不清。Shh信号在肝脏上皮间质转化（EMT）中具有明确的作用，核转录因子Gli直接调控靶基因的转录表达，Snail是其靶基因，也是EMT的关键调控因子。本课题拟研究Shh信号通路核转录因子Gli1/Gli2在胆道闭锁肝脏上皮间充质化（EMT）中的调控作用。.方法：选择经手术证实为胆道闭锁III型患儿的肝脏活检组织标本，年龄为1-3个月，以相同年龄段无消化道畸形死亡新生儿尸检肝脏组织作为正常对照。应用Real-time PCR及Western-blot法检测肝脏组织中Gli1/Gli2、EMT关键调控因子Snail/Slug及EMT特征性细胞因子的表达。然后在肝内胆管上皮细胞mIBEC中应用RNA干扰技术研究Shh信号通路关键转录因子Gli1/Gli2表达对 EMT过程的调控作用。再应用恒河猴轮状病毒建立胆道闭锁模型鼠，构建过表达及沉默Gli1/Gli2基因的腺病毒载体及慢病毒载体并制备病毒，在胆道闭锁模型阳性鼠体内检测Snail/Slug基因及EMT特征性细胞因子的表达改变。.结果：与对照组比较，胆道闭锁患儿肝脏组织Gli2、Snail、Vimentin、α-SMA表达明显升高（P<0.05），E-cadherin表达明显降低（P<0.05），Gli1、Slug表达无明显变化（P>0.05）。应用RNA干扰技术沉默Gli2基因后，肝内胆管上皮细胞mIBEC中Snail、Vimentin、α-SMA表达明显减低（P<0.05），E-cadherin表达升高（P<0.05）；过表达Gli2基因后，Snail、Vimentin、α-SMA表达明显升高（P<0.05），E-cadherin表达降低（P<0.05）。同样，应用RNA干扰技术在胆道闭锁阳性鼠体内过表达Gli2后，小鼠肝脏组织Snail、Vimentin表达明显升高（P<0.05），E-cadherin表达降低（P<0.05）；沉默Gli2表达后，出现相反变化。沉默及过表达Gli1基因在肝内胆管细胞及胆道闭锁模型小鼠肝脏未见明确Snail及EMT相关因子表达变化趋势。.结论：Shh信号通路在胆道闭锁肝纤维化中发挥的重要作用，信号通路关键转录因子Gli2可显著调控胆管上皮细胞EMT过程。