CCR2阳性单核/巨噬细胞促进脑缺血长期神经功能恢复的机制及其药物干预研究

Ischemic stroke induced inflammation is a main focus in current research. Regulating macrophage will facilitate inflammation recovery, but it is still unknown in long term recovery after ischemic stroke. Our previous research showed that CCR2 expressing monocyte/macrophage aggravated infarct size in acute stage, while sustained survival and promoted long term recovery after ischemic stroke. The underline mechanism may be related to M1/M2 macrophage polarization. In this project, we will set up ischemic stroke model in CCR2 knockout, and wild type mice. Long term survival, behavior and memory test will be observed to verify the effect of CCR2 expressing monocyte/macrophage on long term recovery from ischemic stroke. We will use flow cytometry analysis, sorting, and immunofluorescence to study M1/M2 macrophage polarization in different time after ischemic stroke. The results will indicate the relationship between M1/M2 macrophage ratio and proinflammatory/tissue repair conversion. We will apply M2 macrophage adoptive transfer to explore the function of M2 macrophage after ischemic stroke. At last, we will use some medicine, such as IL-4 complex, XQ-1H, to approve the signal pathway IL-4→pSTAT6→PPARγ which will stimulate M2 macrophage polarization both in vivo and in vitro. The expectant results of this project will reveal the potential target on long term recovery from ischemic stroke, and will give evidence to M2 macrophage polarization target medicine on ischemic stroke treatment.

脑缺血诱导的炎症反应是研究热点，调控巨噬细胞极化会影响炎症转归，但在脑缺血长期神经功能恢复中的机制尚未阐明。前期研究表明，CCR2阳性单核/巨噬细胞在脑缺血急性期加重梗死，然而长期却维持动物存活、促进神经功能恢复，其机制与巨噬细胞M1/M2型极化有关。本项目旨在用CCR2基因敲除小鼠及其CCR2抑制剂，阐释CCR2阳性单核/巨噬细胞对脑缺血长期恢复的影响；应用流式细胞分选、免疫荧光等技术研究脑缺血后巨噬细胞极化过程，揭示M1/M2比例的动态变化与促炎/修复的内在关系；用M2巨噬细胞过继移植，阐明脑缺血后M2巨噬细胞促进神经修复的功能；用IL-4 complex、XQ-1H等药物干预，探讨IL-4→pSTAT6→PPARγ信号通路调控M2巨噬细胞极化及其促进脑缺血长期恢复的分子机制。研究结果将揭示脑缺血后长期神经功能恢复的潜在治疗靶点，并为调控M2巨噬细胞极化的相关药物研究建立平台。

脑缺血后CCR2阳性单核细胞向脑组织迁移浸润并分化为巨噬细胞，加剧免疫炎症反应的同时也会促进神经功能修复，然而影响时程及其机制并未明确。.本项目首先应用CCR2 KO小鼠，发现脑缺血后单核细胞向缺血脑组织迁移是CCR2依赖性的，CCR2 KO小鼠脑缺血后脑组织单核细胞数目较WT小鼠显著减少；在此基础上，应用CCR2 KO小鼠及CCR2抑制剂，明确CCR2阳性单核/巨噬细胞在脑缺血后急性期加重损伤，而在后期促进神经功能修复的双刃剑作用。为了阐明机制，应用Real time PCR、流式细胞及其分选技术，揭示脑缺血后早期CCR2阳性单核/巨噬细胞主要向致炎（M1）型巨噬细胞极化，而在恢复期则主要向抑炎（M2）型巨噬细胞极化。因此，CCR2阳性单核细胞在脑缺血后早期加重损伤，而在后期却是神经功能修复不可缺少的重要细胞。M2巨噬细胞、TGF-β在脑缺血恢复期静脉注射，都能明显改善CCR2小鼠神经功能修复和动物生存率，进一步证实了CCR2阳性单核/巨噬细胞对脑缺血功能修复的重要作用。最后，阐明了抗脑缺血新药XQ-1H通过PPARγ信号通路促进M2巨噬细胞极化、减轻脑缺血损伤的分子机制。.研究结果揭示CCR2阳性单核/巨噬细胞在脑缺血后具有双刃剑的作用，调控单核/巨噬细胞迁移及其极化对脑缺血的预后有重要意义。本项目已按照预期完成极化任务书的研究内容，发表论文7篇，其中SCI论文5篇，培养博士研究生3名，硕士研究生3名。