黄芪汤调控肝祖细胞NUMB/NOTCH信号通路抗胆汁性肝纤维化的机制研究

Abnormal proliferation of bile duct is a key pathological process in cholestatic liver fibrosis and Notch signal activation is the major signal transduction mechanism in this process. So far there are no an effective therapeutics for cholestatic liver fibrosis which is an urgent scientific problem need to be resolved. Based on our previous research, Notch signaling promotes cholestatic liver fibrogenesis, as well as effects of Huangqi decoction on Notch signaling and its negative regulator Numb, we hypothesized that " Numb might play an important role in preventing cholestatic liver fibrogenesis. Huangqi decoction could prevent cholestatic liver fibrosis by inhibiting activation of Notch signaling in hepatic progenitor cells; While Numb, an inhibitor of Notch signaling, might be a key target of this decoction". Our study will focus on the association between Numb expression in hepatic progenitor cells and cholestatic liver fibrogenesis, as well as anti-fibrotic effects of Huangqi decoction on regulating Numb expression. The fetal hepatic progenitor cells (HPCs) are isolated from GFP-transgene Sprague Dawley (SD) rats, and the HPCs with Numb over expression or Numb-knockdown by genetic modification will be transplanted into livers of common bile duct ligation (BDL) rats respectively; and these rats are treated with Huangqi decoction at the same time. By combining results with in vitro experiments, the relationship between Numb expression and liver fibrosis, and effect of Huangqi Decoction against cholestatic liver fibrosis through regulating Numb expression will be revealed. The project will elucidate the molecular mechanism of Huangqi decoction against cholestatic liver fibrosis and provide scientific evidence for its clinical application.

肝内胆管异常增生是胆汁性肝纤维化发生的关键病理环节，Notch信号通路活化是其关键信号转导机制，但目前尚缺乏有效的干预措施，成为亟需解决的关键科学问题。本课题基于前期对Notch信号促进胆汁性肝纤维化发生，以及黄芪汤对Notch信号及其负调控因子Numb影响的研究，提出“Numb对胆汁性肝纤维化的发生可能具有重要的干预作用，黄芪汤干预胆汁性肝纤维化的作用机制与抑制肝祖细胞Notch信号通路的活化有关，而Numb可能是其关键作用靶点”假说。以肝祖细胞Numb基因水平与胆汁性肝纤维化的发生及其对黄芪汤抗纤维化作用的影响为主要研究内容，拟采用胎肝祖细胞基因改造移植技术，分离绿色荧光蛋白转基因SD大鼠胎肝祖细胞，使Numb基因过表达或表达下降后植入胆总管结扎SD大鼠肝脏，并予黄芪汤干预，结合体外实验，揭示Numb与肝纤维化的关系以及黄芪汤治疗胆汁性肝纤维化的效应机制，为该方的临床应用提供科学依据。

背景与目的：前期研究表明黄芪汤可提高胆汁性肝纤维化（cholestatic liver fibrosis,CLF）肝脏Numb的表达水平，抑制Notch信号激活，抑制肝干细胞向胆管上皮细胞分化，进而发挥抗肝纤维化作用。但肝干细胞Numb基因在CLF发病中的重要作用、以及黄芪汤抗CLF作用与Numb基因之间的关系尚不清楚。.方法：建立胆管结扎大鼠CLF模型：（1）观察阻断Notch信号通路对肝干细胞分化及CLF进展的影响；（2）观察骨髓间充质干细胞（BMSCs，外源性肝干细胞）Numb敲减（BMSCNumb-KD）对CLF进展及其对黄芪汤抗CLF作用的影响；（3）观察BMSCs Numb过表达（BMSCNumb-OE）对CLF进展及其对黄芪汤抗CLF作用的影响；（4）以腺相关病毒（AAV）为载体，观察体内Numb过表达对CLF的影响。此外，本研究还检测了PBC患者肝脏Numb蛋白的表达水平。.结果：在胆管结扎大鼠CLF：（1）新生胆管上皮细胞主要来源于肝干细胞，阻断Notch信号通路可显著抑制肝干细胞向胆管上皮细胞分化以及CLF进展；（2）黄芪汤可显著抑制Notch信号通路的活化，抑制肝干细胞向胆管上皮细胞分化，抑制CLF进展；（3）BMSCs Numb基因敲减移植后，可向胆管上皮细胞分化，可促进Notch信号通路活化，进而促进CLF发展。（4）BMSCs Numb基因敲减移植后再予以黄芪汤干预，显著降低了黄芪汤抗CLF的作用；（5）BMSCs Numb基因过表达移植后，可向肝实质细胞分化而不是向胆管上皮细胞分化，显著抑制Notch信号活化，进而抑制CLF进展，但BMSCs Numb基因过表达移植并未进一步提高黄芪汤抗CLF的作用。（6）以AAV为载体的体内Numb基因过表达可显著改善肝功能，抑制Notch信号活化及胆管反应，进而抑制CLF进展。此外，PBC患者肝脏Numb蛋白显著低于健康对照组。.结论:（1）干细胞是CLF新生胆管上皮细胞的主要来源，阻断Notch信号通路可抑制这一病理改变。（2）干细胞Numb在CLF的发展中发挥关键作用，Numb敲减促进CLF的进展；而Numb过表达则可显著抑制这一病理进程。（3）黄芪汤抗CLF的作用机制与提高干细胞Numb的水平有关。不仅为CLF的治疗提供了新思路，也为以Numb为靶点发掘黄芪汤抗CLF的有效成分奠定了基础。