Notch-RBPJ调控肝祖细胞介导的肝内胆管再生与重建的分子机制研究

Liver progenitor cells are bipotentiality cells that can differentiate to hepatocytes and biliary epithelial cells.In the setting of liver injury and diseases, liver progenitor cells play an improtant role in the liver regeneration and repair process. The coordinated proliferation of hepatocytes and biliary epithelial cells to efficiently form intrahepatic biliary ducts is pivotal to the structure and funciton of liver regeneration. Notch-RBPJ is very important in the maintance of hepatic progenitor cells homeostasis, proliferation, differentiation, especially in bile duct injury and regeneration. In previous study we found that liver specific RPBJ knockout would lead to bile duct develop abnormalities in perinatal and cholestasis. Therefore, we hypothesize that Notch-RBPJ plays a significant role in the progress in liver disease by regulating the proliferation and differentiation of liver progenitor cells, bile duct rebuild and mediate the bile duct injury and cholestasis. To test this hypothesis, we will apply genetic mice models, cre-loxp system for lineage tracing by Rosa 26 gene report and RBPJ gene knockout especially in liver. We will explore the mechanism of Notch-RBPJ pathway in regulating liver progenitor cells and bile duct reconstruction, and analyze the effect of the pathway in regulating of target genes. It would not help us to understand the machinery in liver regeneration, but also provide the evidence for therapeutic target and pathogenesis of bile duct reconstruction and regeneration.

肝祖细胞作为双潜能肝干细胞，在肝脏再生中发挥重要作用，进一步分化为肝细胞和胆管细胞。两者的协调增殖及肝内胆管通道的有效形成，是肝脏组织结构和功能恢复的重要保证。Notch-RBPJ通路对维持肝祖细胞的稳态，调控其分化增殖，尤其对胆管的再生重建、损伤至关重要。我们前期研究发现肝脏特异性敲除RBPJ基因的小鼠，在围生产期存在胆管发育异常，出现胆汁淤积。为此，我们提出假说：Notch-RBPJ信号通路通过调控肝祖细胞增殖分化，介导胆管再生与重建，是肝脏再生的关键机制。为验证这一假说，我们拟通过基因敲除小鼠的体内模型和体外细胞实验，采用谱系示踪技术在小鼠中转入Rosa26报告基因来示踪肝祖细胞；Cre-loxP技术在肝脏特异性敲除RBPJ基因等手段，探讨该通路对肝祖细胞的增殖分化、胆管再生与重建的作用，并明确其下游靶基因调控机制。为探索肝脏再生，尤其是胆管再生与重建提供新的靶点及分子机制。

成功构建带报告基因的先天性肝脏特异性敲除RBPJ基因小鼠，即Alb Cre+;RBPJ flox/flox；Rosa26mTm/G+，Rosa26laz/+基因型小鼠。.1．发现Alb Cre+;RBPJ flox/flox基因型小鼠在围生产期存在胆管发育异常，出现肝内胆汁淤积。.2．成功构建带报告基因的可诱导的肝脏特异性敲除RBPJ基因小鼠，Mx1 Cre+;RBPJ flox/flox;Rosa26mTm/G+基因型小鼠，该小鼠可以被poly（I:C）诱导敲除RBPJ基因。.3．我们发现在正常成年小鼠中敲除RBPJ基因后，会导致肝脏再生功能障碍，具体表现为胆管再生障碍，胆汁淤积。.4．通过基因示踪技术，在肝脏再生模型中，我们发现再生的胆管细胞部分来源于带标记的肝祖细胞，而RBPJ基因敲除后，这种现象消失。.5．在正常成年小鼠肝脏胆汁淤积性肝硬化模型中，敲除RBPJ基因后，会导致小鼠对该疾病的易感性增高，疾病进展快，死亡率明显上升。.6．通过体外实验我们发现，Notch-RBPJ信号直接影响肝祖细胞，肝细胞，和胆管细胞的生物学活性，Notch-RBPJ信号影响肝祖细胞的增殖、分化和凋亡。.7．Notch-RBPJ对肝祖细胞的增殖分化是通过Hippo信号通路来实现的，可以影响Hippo-YAP基因的入核。. 综上所述，Notch-RBPJ在肝脏的发育、再生以及肝脏胆汁淤积性肝病的进展中起重要作用，通过对肝祖细胞增殖分化的调控作用，影响胆管上皮细胞的功能，胆管树的再生来影响肝脏再生进程也通过对胆管形态和功能修复的调控作用参与胆汁淤积性肝病的进展。这些都为我们进一步研究有关肝脏疾病尤其是胆汁淤积性肝病的诊断和治疗奠定了重要的理论依据，提供了新的靶点。