CXCL4/CXCR2募集造血干细胞来源巨噬细胞调控肝星状细胞促进肝纤维化逆转/消退的作用机制

Macrophages in the liver are the key regulatory cells for the reversion/regression of liver fibrosis. Our preliminary data revealed that there was a group of metalloproteinases-expressing macrophages directly derived from hematopoietic stem cells in regressing phase of liver fibrosis. Yet, it is still unclear how these cells were recruited to the liver and what functions of these cells were. We hypothesize that the macrophages, which were directly derived from hematopoietic stem cells, were recruited to the liver via CXCL4/CXCR2 chemokine signaling pathway and promoted fibrosis regression through inducing apoptosis/inactivation of hepatic stellate cells. To test this hypothesis, hematopoietic stem cell-lineage tracing mice and conditional-depletion mice will be used to confirm that the macrophages directly derived from hematopoietic stem cells participate in the regression of liver fibrosis and their number are correlated with the speed of regression. Secondly, the antagonists of CXCL4 and/or CXCR2 will be used to block the recruitment of hematopoietic stem cell-derived macrophages in order to reveal the recruitment mechanism of these cells. Finally, in vitro coculture system will be used to reveal the apoptosis/inactivation-inducing effects of hematopoietic stem cell-derived macrophages on hepatic stellate cells. And these effects will be confirmed by hematopoietic stem cell-lineage tracing mice, conditional-depletion mice, CXCL4 and/or CXCR2 recruitment blocking mice. We hope this study will provide insight into the understanding of the behavior and function of the macrophages directly derived from hematopoietic stem cells in regressing liver fibrosis.

肝脏巨噬细胞是决定纤维化进展与逆转/消退的关键调节细胞。我们发现在肝纤维化逆转/消退阶段，肝组织募集由造血干细胞直接来源的、高表达MMP的巨噬细胞，但其募集机制和发挥的功能尚不明确。推测趋化因子CXCL4/CXCR2募集的、造血干细胞直接起源的巨噬细胞通过调控肝星状细胞而促进纤维化消退。首先利用造血干细胞示踪和条件性清除转基因小鼠，通过募集更多或清除造血干细胞直接来源的巨噬细胞，以明确其数量消长与纤维化逆转/消退的关系；然后应用CXCL4与CXCR2的特异性拮抗剂干预其募集过程，阐明其募集到肝脏中的分子机制；最后应用体外共培养体系明确其诱导肝星状细胞凋亡/去活化的功能，并利用造血干细胞示踪和条件性清除转基因小鼠、干预募集过程小鼠的肝组织免疫荧光染色验证其诱导肝星状细胞凋亡/去活化以恢复静息状态的功能。期望本研究能为认识造血干细胞起源的巨噬细胞在肝纤维化逆转/消退中的行为与功能提供实验依据。

背景：严重危害我国人民健康。由于仅仅去除病因不能完全逆转纤维化/硬化，故仍需进一步探讨促进肝纤维化逆转/消散的有效途径。肝脏巨噬细胞是决定肝纤维化进展与逆转/消退的关键性调节细胞，本课题组的前期研究发现提示，造血干细胞直接来源的Ly6clow巨噬细胞参与调控了纤维化逆转/消退过程，但其募集到肝脏来的机制尚且不明。结合既往研究基础和我们的前期发现我们提出趋化因子CXCL4/CXCR2途径募集来的、肝血干细胞直接起源的巨噬细胞通过诱导感性状细胞凋亡或去活化以重新恢复静息状态进而促进肝纤维化逆转/消退。.主要研究内容：(1). 利用造血干细胞示踪和条件性清除转基因小鼠，明确造血干细胞来源的巨噬细胞在 肝脏纤维化消退过程中的作用。(2). 利用 CXCL4 与 CXCR2 的拮抗剂，阐明干预造血干细胞来源巨噬细胞的募集过程 对纤维消退的影响。(3).利用造血干细胞来源巨噬细胞与肝星状细胞的共培养体系，探讨造血干细胞来源巨 噬细胞促纤维消散作用的分子机制。.重要结果：(1). Ly6clow造血干细胞来源的巨噬细胞在肝纤维化的消退过程中数量显著增多，分泌MMP等促进纤维化消散的细胞因子。(2). CXCR2 的拮抗剂，抑制了Ly6clow造血干细胞来源的巨噬细胞向肝脏募集，加重纤维化。(3).利用造血干细胞来源巨噬细胞与肝星状细胞的共培养体系，我们发现造血干细胞来源巨 噬细胞通过诱导肝星状细胞凋亡发挥促纤维消散作用。.通过本研究，我们明确了在肝纤维化的逆转/消散期，Ly6clow巨噬细胞通过诱导肝星状细胞凋亡发挥促进肝纤维化的逆转和消散的作用。