M2r巨噬细胞对实验性脑梗死的免疫调节机制探讨

Cerebral infarction (CI) is of high mortality and disability, but with limited effective therapy. Many researches have indecated that immunologic mechanisms play an important role in the pathogenesis of CI. Immunomodulatory therapies have been proved effective in animal models of CI, including inducing regulatory T cell, enhancing anti-inflammatory cytokines, suppressing pro-inflammatory cytokines, and inhibiting the activation of microglia/macrophage, etc. Researches have showed that M2r macrophage has the immunomodulatory capacity mentioned above, but whether it can be used for the therapy of cerebral infarction and the immunologic mechanisms involved have not been confirmed. Our preliminary research has demonstrated that M2r macrophage can not only reduce the infarct volume and improve neurologic score of middle cerebral artery occlusion (MCAO) mice, but also down-regulate some inflammatory cytokines in the ischemic cerebral tissue, including TNF-α, IL-1β, and IL-17. Besides, M2r macrophage up-regulated the proportion of peripheral regulatory T cells. Based on these evidences, we propose that M2r macrophage might ameliorate CI by its immunomodulatory capacity, including secreting anti-inflammatory cytokines, suppressing pro-inflammatory cytokines, inducing regulatory T cell, and inhibiting the activation of inflammatory cells and their infiltration into the ischemic cerebral tissue. The object of our project is MCAO mice, as the animal model of CI. The techniques of immunology, molecular, pathology, and cell biology would be used in the project. The aims of our project are to study the effects of M2r macrophage on CI and its immunologic mechanisms, and to provide scientific evidenced for using M2r macrophage as a therapy to CI.

脑梗死具有很高的致残率和致死率，但特效的治疗手段非常有限。免疫机制在脑梗死的发病中起着关键作用。诱导Treg细胞产生、增强抗炎因子、抑制促炎因子和小胶质-巨噬细胞活化等免疫调节治疗在脑梗死动物模型已被证实有效。M2r巨噬细胞具有上述作用，那么其对脑梗死是否具有治疗作用？其免疫机制是什么？我们的预实验显示M2r巨噬细胞能减少脑梗死小鼠的脑梗死体积及改善神经功能评分，下调脑缺血区TNF-α、IL-1β和IL-17等促炎因子，增加外周血Treg细胞的比例。据此我们提出假设：M2r巨噬细胞在中枢和外周通过分泌抗炎因子、下调促炎因子、诱导Treg细胞的产生、抑制炎症细胞的活化和向脑部浸润等免疫机制发挥对脑梗死的治疗作用。本项目拟以脑梗死的小鼠和细胞模型为研究对象，通过免疫、分子、病理、细胞生物学等方法，阐明M2r巨噬细胞对脑梗死的治疗作用及其中的免疫机制，为应用M2r巨噬细胞治疗脑梗死提供科学依据。

免疫机制在脑梗死的发病中起着关键作用。M2r巨噬细胞具有免疫调节作用，而且预实验显示M2r巨噬细胞能减少脑梗死小鼠的脑梗死体积及改善神经功能评分，下调脑缺血区TNF-α、IL-1β和IL-17等促炎因子，增加外周血Treg细胞的比例。本课题使用M2r巨噬细胞治疗MCAO小鼠，发现脑部的Treg细胞、M2型小胶质细胞增多，γδT、M1小胶质/巨噬细胞减少，MAPK通路主要转录因子的表达及其磷酸化水平均被抑制，Claudin-5水平升高，外周血中的M1型巨噬细胞和γδT细胞减少，其余的免疫细胞无明显差异。采用RNA干扰技术分别抑制M2r巨噬细胞分泌IL-10和TGF-β的能力后，M2r巨噬细胞则不能减少MCAO小鼠的梗死体积。使用MAPK通路抑制剂和CD25抗体后，M2r巨噬细胞也不能减少MCAO小鼠的梗死体积。在体外实验，使用M2r巨噬细胞对OGD-R细胞模型进行干预，能显著减轻神经元的凋亡，MAPK通路关键蛋白及其磷酸化水平被抑制，使用MAPK抑制剂或经RNA干扰后，M2r巨噬细胞治疗组的原代神经元凋亡则与MCAO组的无明显差异。在M2r巨噬细胞治疗组，小胶质细胞的TNF-α、IL-1β分泌减少，向M1亚型分化减少，向M2亚型分化增多，MAPK通路关键蛋白及其磷酸化水平被抑制，在RNA干扰组和MAPK抑制剂组，则与OGD-R组无显著差异。上述结果表明，M2r巨噬细胞在中枢和外周通过分泌抗炎因子、下调促炎因子、诱导Treg细胞的产生、抑制炎症细胞的活化和向脑部浸润等免疫机制发挥对脑梗死的治疗作用，为应用M2r巨噬细胞治疗脑梗死提供了科学依据。