肝纤维化形成过程中Notch信号通路在肝窦毛细血管化和纤维间隔血管化发生中的作用

Sinusoidal capillarization and fibrous septa vascularization are pivitol elements on the progress of liver fibrosis and its reversal. Our preliminary study demonstrated that Notch signalings are the main pathway enhancing with the increased extent of fibrosis by microarray analysis on chronic hepatitis B patients with different stages of liver fibrosis showed that inhibition of Notch signalings could decrease the the extent of sinusoidal capillarization of sinusoidal epithelial cells. In this study, we will establish the chronic or acute liver fibrosis model in mice induced by injection of carbon tetrachloride or bile duct ligation. we also will establish the mice model of spontaneous reversal of liver fibrosis by stopping the injection of carbon tetrachloride. We aim to investigate the dynamic changes of hepatic vasular system including sinusoidal capillarization and fibrous septa vascularization in the liver fibrogenesis and regression by computerized 3D remodelling techniques. We also want to explore the role of Notch signaling in the occurence and progression of sinusoidal capillarization and fibrous septa vascularization in mice by in vivo and in vitro experimens. Futhermore, we plan to confirm and explore the key mechnisms on hepatic vascular remodelling in the process of liver fibrosis on the basis of liver histological analysis in chronic hepatitis B patients. Therefore, we aim to provide clues for the formation, prevention and treatment of liver fibrosis.

肝窦毛细血管化和肝内纤维间隔血管重构是影响肝纤维化进展及能否逆转的关键，我们前期对慢性乙肝患者基因芯片研究发现Notch信号通路随着纤维化进展明显增强，抑制Notch信号可降低窦内皮细胞毛细血管化。本研究建立四氯化碳肝纤维化及撤药后肝纤维化自发性逆转、胆管结扎诱导的急慢性肝损伤小鼠模型，通过计算机3D成像技术，观察急慢性肝纤维化形成过程及肝纤维化逆转过程中肝窦和纤维间隔新生血管形成及改建过程；分别通过肝脏选择性基因敲除小鼠和窦内皮细胞的Notch受体，观察不同信号活化和抑制后肝纤维化间隔血管重构及肝窦毛细胞血管化的变化；细胞实验分析Notch信号对活化肝星状细胞促进肝窦毛细血管化及增强内皮细胞血管形成能力影响；临床验证和分析慢性乙肝患者肝组织纤维间隔血管化和肝窦毛细血管化发生过程和发生机制，为肝纤维化发病机制和防治提供实验和临床依据。

肝窦内皮细胞的毛细血管化出现在肝纤维化的起始阶段，被认为是肝纤维化的门控事件。DLL4，作为 Notch 信号通路的配体之一，主要表达于血管内皮细胞，在维持肝窦稳态中具有重要作用。应用免疫组织化学及免疫荧光技术判断HBV所致人肝硬化组织及CCl4诱导的小鼠肝纤维化组织DLL4的表达及分布。利用免疫组织化学技术动态观察CCl4诱导的肝纤维化小鼠肝窦内皮细胞毛细血管化的标志分子CD31、vWF 的表达，应用扫描电镜动态观察肝窦内皮细胞窗孔的变化，透射电镜动态观察 肝窦内皮下细胞基底膜的形成。RT-PCR 和Western blot检测肝窦内皮细胞DLL4的表达，Notch 信号通路的激活情况及细胞基底膜组份Ⅰ、Ⅲ、Ⅳ型胶原蛋白，层黏连蛋白，纤黏连蛋白的表达。构建DLL4过表达和敲减的人肝窦内皮细胞 (TMNK-1)慢病毒稳定转染细胞株，RNA-sequence分析DLL4过表达引起肝窦内皮细胞毛细血管化的机制。利用Transwell实验及人肝窦内皮细胞与星 状细胞的共培养探索DLL4对肝内血管阻力的影响。低氧培养明确低氧诱导因 子-1α是否能诱导肝窦内皮细胞DLL4的表达。最后构建针对小鼠DLL4的siRNA，通过尾静脉注射观察其对CCl4造模小鼠肝窦内皮细胞毛细血管化的缓解及肝纤维化的治疗作用DLL4 介导的 Notch信号可促进肝窦内皮细胞的毛细血管化。研究结果显示DLL4 在肝纤维化肝脏血管病理性改建中发挥重要作用。我们的研究主要探讨 DLL4在肝窦内皮细胞毛细血管化中的作用，及其在肝纤维化发生、进展中的角色。进一步 揭示肝纤维化的发病机制，为肝纤维化的治疗提供新思路。项目成果在BBA-Molecular Cell Research上发表论文一篇。