胆道闭锁肝纤维化上皮间充质转化及TGFβ-BMP-Smad信号通路调控机制的研究

Biliary atresia (BA) is a progressive fibro-obliterative disorder that results in an obstructive cholangiopathy leading to cirrhosis and end-stage liver disease in infants. Previously, we demonstrated that epithelium- mesenchymal tansition (EMT) was involved in the progressive fibrosis and eventually disappearing of biliary tract, on which TGF-BMP-Smad signalling pathway played important roles, and development genes SHH and Notch were involved. In this study,we will develop a cell strain of mice biliary epithelium cell (BEC), the pivotal role of TGFβ in inducing EMT will be observed. shRNA interference technique will be employed at different steps of TGFβ-BMP-Smad pathway; by knocking-down CCN2 (the downstream target gene of TGF-β1) and knocking-up ALK3 (the specific receptor gene of BMP7) in BEC we will study the regulating role of RNAi on EMT in vitro. MMU18006 rotavirus was inoculated into Balb/c mice t produce BA animal models. In vivo studies will be performed to confirm the in vitro results, and to confirm the anti-fibrosis effects of RNAi. Through this study, we aimed to understand more about the mechanism of progressive cholangiopathy and hepatic fibrosis of BA, and the effect of RNAi, and to find a new target for further therapy.

胆道闭锁（BA）是婴儿胆管进行性纤维化、消失、最终导致终末期肝硬化的一种疾病，是儿童期肝移植的主要原因之一。前期研究中我们发现上皮间充质转化（EMT）机制参与了胆管的进行性纤维化并最终导致肝内外胆树的消失，其中TGFβ-BMP-Smad信号通路发挥重要作用，发育基因SHH、Notch等参与这一过程；本研究拟在前期基础上体外分离培养小鼠胆管上皮细胞，观察外源性TGFβ诱导胆管上皮细胞发生EMT的作用及机制，应用shRNA在TGFβ-BMP-Smad信号通路不同环节进行RNA干扰，特异性沉默TGFβ下游基因CCN2、上调BMP受体基因ALK3，观察对体外培养胆管上皮细胞EMT的逆转作用，应用轮状病毒MMU18006致畸新生小鼠，产生BA小鼠动物模型，将携带目的基因shRNA的慢病毒载体转入活体动物体内，观察RNA干扰对活体小鼠胆管及肝实质纤维化的影响，探讨其作用机制，为新治疗方法的提出提供依据

胆道闭锁是导致新生儿及小婴儿梗阻性黄疸的主要原因之一，不经治疗很快发展为肝硬化、死亡，即使成功行肝门空肠吻合术，术后自体肝存活率也仅能达到30-40%，因而胆道闭锁已经成为儿童肝移植的主要病因。胆道闭锁发病原因并不清楚，对胆道闭锁进一步深入研究对于阐明该病的发病机制、改善预后至关重要。本研究给新生小鼠腹腔内注射轮状病毒能成功模拟人类胆道闭锁，注药后7天开始胆管发生部分纤维化，10天-14天逐渐消失，TGF-β信号通路诱导的上皮间充质转化在胆管上皮的纤维化、消失过程中发挥作用，TGF-β信号通路重要分子CTGF和BMP7在胆道闭锁患儿和胆道闭锁小鼠肝脏组织中表达均明显升高。应用TGF-β诱导体外培养的胆管上皮细胞可使上皮细胞表达间充值特异性标记物α-SMA，而且细胞形态及迁移能力发生改变，在胆管发生上述异常变化的过程中Toll样受体(TLR)信号通路发挥重要作用，尤其是TLR7,在胆管逐渐消失的过程开始阶段明显升高，因而可能可以作为胆管损伤不可逆标记物。