肝纤维化中骨髓来源的纤维细胞在肝脏募集与分化的作用研究

Activation and proliferation of hepatic myofibroblasts are required for the development of liver fibrosis. One of the most exciting new concepts in fibrosis is the potential contribution of Bone Marrow (BM) derived fibrocytes to liver fibrosis, that are CD45 and Type I collagen expressing cells. It has been confirmed that BM-derived fibrocytes contribute 4-6% of all collagen expressing cells (myofibroblasts) in the fibrotic liver. Fibrocytes can differentiate into multiple cell types under different conditions, but the differentiation of recruited BM-derived fibrocytes in injuried liver remains unclear. Our previous studies found that Serum Amyloid P (SAP), which is the natural inhibitor of fibrocytes differentiation, could significantly attenuate liver fibrosis by 45%, indicating the important role of fibrocytes in the liver fibrogenesis. Accordingly, we hyposithesied that fibrocytes are mobilized from bone marrow to the liver in response to injury to generate multiple cell types that stimulate fibrosis, and that targeting fibrocyes in the setting of liver fibrosis will improve fibrosis. In this study, we will first use inducible genetic ablation of fibrocytes to determine the role of fibrocytes in experimental hepatic fibrosis. Ablation of fibrocytes throughout the duration or at the onset of liver injury was carried out in carbon tetrocloride (CCl4) and bile duct ligation (BDL) induced liver fibrosis. Secondly, we will use pharmacological inhibition of fibrocytes-SAP to determine the important role of fibrocytes in the pathogenesis of liver fibrosis. SAP will be injected into both CCl4 and BDL induced liver fibrosis mice model and the recruitment of fibrocytes will be observed in CCl4-treated Col-GFP→wt mice by GFP expression. The inhibition of fibrocytes recruitment will be observed by GFP expression. Finally, to gain greater insight into mechanisms by which a relatively small number of fibrocytes can substantially contribute to fibrosis, we develop a cell fate mapping approach to determine fibrocyte functions. The differentiation of fibrocytes into myofibroblasts, macrophages, and dendritic cells will be detected, and their characteristics will be compared with that of resident macrophages and dendritic cells in liver. The results of our studies will give greater insight into mechanisms underlying the role of fibrocytes in liver fibrosis and validate fibrocytes as a potential therapeutic target.

活化的肌成纤维细胞（myofibroblasts, MF）在肝纤维化进程中发挥重要作用,骨髓来源的纤维细胞可以活化为MF参与纤维化进程，其在体内外不同环境中具有多相细胞分化潜能，但在受损肝脏中的分化尚未得到证实。前期研究发现：抑制纤维细胞分化的血清淀粉样物质P（SAP），具有明显的抗肝纤维化作用,提示纤维细胞在受损肝脏中可能发挥重要促纤维化作用，与其具有的多相分化潜能相关。本课题拟利用四氯化碳及胆管结扎诱导的小鼠肝纤维化模型，首先在肝纤维化全程及纤维细胞向肝脏募集高峰阶段条件性敲除骨髓来源的纤维细胞，观察肝纤维化改善情况；其次在纤维化起始阶段及已发生阶段利用化合物SAP进行干预，同时明确纤维化的改善与纤维细胞向肝脏募集相关；最后通过细胞示踪方法，研究肝脏损伤过程中骨髓来源的纤维细胞在肝脏中分化为Kupffer细胞和树突状细胞的潜能，为全面了解纤维细胞在肝纤维化进展过程中的作用提供实验依据。

Pilling等首先证实血清淀粉样物质P（SAP, serum amyloid P）——天然的抑制纤维细胞分化的化合物，在争光霉素诱导的小鼠及大鼠肺纤维化模型中可以显著抑制纤维细胞在纤维化肺脏的聚集及活化，进而减少肺脏胶原沉积。鉴于纤维细胞在肝纤维化过程中具有的转化为肌成纤维细胞的特性，本课题进行了化合物SAP在不同病理损伤情况下，抗肝纤维化作用及机制探讨研究。首先证实了SAP具有一定的抗CCl4及BDL诱导的小鼠肝纤维化作用，即将一定剂量的SAP经腹腔注入经CCl4或者BDL诱导的小鼠肝纤维化模型体内，造模后6周或者术后17天进行模型组与SAP干预组小鼠肝脏的病理学检测,发现SAP干预组小鼠肝脏纤维化程度相比于模型组显著降低,天狼猩红染色证实与模型组相比，胶原沉积降低40%以上；肝脏巨噬细胞浸润及活化程度均降低50%以上，结果表明SAP具有明显的抗肝纤维化作用。其次，通过GFP标记骨髓细胞的骨髓嵌合小鼠证实，SAP具有的抗肝纤维化作用与纤维细胞在肝脏的募集减少相关。最后，证实了SAP对已发生肝纤维化小鼠的抗肝纤维化作用：即利用CCl4及BDL诱导的肝纤维化模型小鼠，于CCl4刺激后的第10天后开始每周两次SAP干预，观察SAP抗肝纤维化作用。SAP对模型组小鼠肝脏胶原沉积起到了有效的抑制作用。.在本研究中我们应用化学合成的抑制纤维细胞分化的化合物的SAP,证实其不仅其通过预防性给药的方式对CCl4及BDL诱导的肝纤维化模型小鼠起到了有效的抗纤维化作用，同时通过治疗性给药的方式证实其对已经形成的CCl4及BDL诱导的肝纤维化模型小鼠起到有效的抗肝纤维化效果。小鼠体内实验证实，SAP的抗肝纤维化作用与肝脏巨噬细胞的募集与活化受抑有关，同时也与SAP对纤维细胞向肝脏募集的抑制作用相关。上述实验结果为SAP作为临床潜在的抗肝纤维化药物提供了一定的研究基础。