疏肝健脾活血方调控VEGF-Notch信号通路抑制肝纤维化过程中血管新生的作用机制研究

Liver fibrosis is secondary to various forms of chronic liver injury after the tissue repair response, will develop into liver cirrhosis and liver cancer without effective treatment. In the process of liver fibrosis is a clear pathological angiogenesis and reconstruction, hindered the reversal and recovery of liver fibrosis. Angiogenesis and its signal transduction system have become potential targets of liver fibrosis treatment. Studies have found that VEGF and Notch signaling pathways can accommodate each other mutual influence angiogenesis, but its mechanism is unclear in the angiogenesis of hepatic fibrosis. This topic with VEGF - Notch signaling pathway in the liver fibrosis process the mutual relations and the role of angiogenesis as the breakthrough point, dynamic observation in vivo rat liver fibrosis formation in the process of VEGF, and the interaction between Notch signaling pathway and Suganjianpihuoxue repice The intervention of the role; In vitro activation and resistance of Notch,transient transfection pcDNA3.1 - VEGFl65 plasmid activation of VEGF, using variety of experimental technology as immunohistochemical, immunofluorescence, transmission electron microscopy (sem) and westernt blot and rt-pcr and so on to analyze the Suganjianpihuoxue repice resisting the specific influence angiogenesis link or targets of hepatic fibrosis, new research ideas and methods will be found to preventiig and treating liver fibrosis with traditional Chinese medicine.

肝纤维化是继发于各种形式慢性肝损伤之后的组织修复反应，若无有效治疗将演变为肝硬化和肝癌。肝纤维化进程中存在明显的病理性血管新生与重构，阻碍了肝纤维化的逆转与恢复。血管新生及其信号调控系统已成为肝纤维化治疗的潜在靶标。研究发现VEGF与Notch信号通路能相互调节共同影响血管新生，但其在肝纤维化血管新生中的机制尚不明确。本课题以VEGF-Notch信号通路在肝纤维化进程血管新生中的相互关系和作用为切入点，体内动态观察大鼠肝纤维化形成过程中VEGF、Notch信号通路的相互作用及疏肝健脾活血方的干预作用；体外分别激活和阻制Notch通路、pcDNA3.1-VEGFl65质粒瞬时转染激活VEGF通路，应用免疫组化、免疫荧光、透射电镜、western blot 和RT-PCR等多种实验技术，观察分析疏肝健脾活血方抗肝纤维化血管新生的具体影响环节或靶点，为中医药防治肝纤维化提供新的研究思路和方法。

肝纤维化是肝细胞发生坏死及炎症刺激时，肝脏中胶原蛋白等细胞外基质(ECM)的增生与降解失去平衡，导致肝脏内纤维结缔组织异常沉积而成，若进一步加重可以向难以逆转的肝硬化转变。肝窦毛细血管化指肝损伤发生后肝窦内皮细胞（LSEC）失去原有窗孔结构、肝星状细胞（HSC）活化分泌相关胶原并沉积形成内皮下基底膜，形似连续毛细血管的过程，是肝纤维化时的结构上表现。近年研究认为肝窦毛细血管化与肝纤维化的形成相互需求、相互促进，因此通过抑制肝窦毛细血管化减缓或逆转肝纤维化的进程成为新的治疗靶点。而血管新生是肝纤维化形成与逆转的关键环节。VEGF是最特异和最关键的血管生成刺激因子，几乎参与了生理性和病理性血管新生的每一步。VEGF与其受体VEGFR结合，通过丝裂原活化蛋白激酶/胞外信号调节激酶信号途径，促进血管新生和肝纤维化。有序且功能完好的血管生成除VEGF信号通路外，需要其他包括Notch信号通路在内的其他信号通路的参与。Notch是VEGF的下游信号通路，VEGF-Notch信号通路间的交互作用对于血管生成具有重要意义。本课题采用四氯化碳成功复制大鼠肝纤维化模型，疏肝健脾活血方由桃仁、桂枝、制大黄、甘草、香附、白术、莪术、鳖甲、参三七、茯苓、丹参、红枣等组成，研究结果表明该方可以改善CCl4大鼠肝纤维化程度，对肝纤维化具有防治作用。体外实验部分采用HSC与LSEC共培养，利用慢病毒转染技术过表达VEGF，激活大鼠HSC内VEGF信号通路；应用Notch信号抑制剂（DAPT）和激活剂（rhNF-κB）,抑制或激活Notch信号。实验结果表明疏肝健脾活血方对VEGF-Notch信号通路间的交互作用具有调控作用。疏肝健脾活血方可升高Notch通路配体DLL4，激活DLL4-Notch信号通路，进而诱导VEGFR2降低，抑制VEGF信号通路，同时能降低Jagged1与VEGF，拮抗血管新生，降低CD31、VWF，缓解肝窦毛细血管化，改善LSEC失窗孔化，促进胶原IV降解，进而逆转肝纤维化的发展。