miR-21调节小胶质细胞、巨噬细胞以及自身免疫脑脊髓炎的作用机制研究

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), is an autoimmune inflammatory diseases, mediated by peripheral inflammatory T cells that infiltrate the CNS, activate resident microglia and recruit macrophage cell. Disease-associated miR-21 is up-regulated in MS and EAE, but its exact mechanism remian unclear. Report shown that no significant changes in T-cell function following miR-21 inhibition in vitro. Our preliminary study found that silencing of miR-21 can significantly inhibits the EAE progression, and reducing the activation of CNS microglia, macrophages. Previous studies have found that PPARγ can inhibit the activation of microglia and promote peripheral monocyte differentiation into anti-inflammatory M2 macrophages. We further found that the inhibition of miR-21 could promote the expression of PPARγ in microglia and macrophages. Therefore, we propose that upregulation of PPARγ by silencing of miR-21 in microglia and macrophages may inhibit the activation of microglia and macrophages and promote the differentiation of monocytes into antiinflammatory M2 macrophages, thereby inhibiting the inflammatory response of CNS. This proposal will focus on study of the roles and mechanisms of miR-21 in regulation of microglia and macrophages and EAE. The success of this study will provide a new way for MS and miR-21 associated autoimmune diseases immunotherapy.

实验性自身免疫脑脊髓炎（EAE）是由T细胞和巨噬细胞以及中枢神经系统（CNS）激活的小胶质细胞介导的自身免疫性炎症疾病。miR-21 与多种疾病相关，在多发性硬化症及其动物模型EAE中表达升高，但其具体作用机制尚不清楚。研究发现体外抑制miR-21对T细胞功能并无影响。而我们研究发现体内沉默miR-21明显抑制EAE发生发展，降低EAE小鼠小胶质细胞和巨噬细胞激活，促进PPARγ表达。已有研究证明PPARγ可抑制小胶质细胞激活并促进抗炎的M2巨噬细胞分化。因此，我们推测沉默miR-21对EAE的调节作用可能通过促进小胶质细胞和巨噬细胞中PPARγ表达，抑制小胶质细胞和巨噬细胞激活并促进单核细胞分化为M2巨噬细胞，从而抑制CNS炎症反应，缓解EAE病情。因此，本项目将在整体、细胞和分子水平综合研究miR-21调节EAE的细胞和分子机制，为多发性硬化症及相关疾病的免疫治疗奠定理论和实验基础。

背景：实验性自身免疫脑脊髓炎（EAE）是由T细胞和巨噬细胞以及中枢神经系统（CNS）激活的小胶质细胞介导的自身免疫性炎症疾病。miR-21 与多种疾病相关，在多发性硬化症及其动物模型EAE中表达升高，但其具体作用机制尚不清楚。..研究内容：1.构建miR-21敲除小鼠模型；2. miR-21 在CD4+表达情况检测；3. 敲低miR-21研究其对EAE发病过程中CNS区炎症细胞浸润和脱髓鞘情况；4. 敲低miR-21如何调控EAE小鼠静息的小胶质细胞；5. 敲低miR-21如何调控体外巨噬细胞激活过程中自噬的发生；6. 敲低miR-21调控外周脾脏和淋巴结中Th17的分化；7.miR-21调节EAE发生的分子靶点。..结果: 1. miR-21在CD4细胞中高表达并与Th17细胞分化具有很强的相关性；2. 敲低miR-21可以明显减低EAE中枢炎症细胞浸润和脱髓鞘情况，降低中枢浸润的Th17细胞比例；3. 敲低miR-21上调EAE小鼠静息的小胶质细胞；4. 敲低miR-21抑制体外巨噬细胞激活过程中自噬的发生；5. 敲低miR-21抑制外周脾脏和淋巴结中Th17的分化；6. miR-21调节EAE发生的分子靶点为Rorγt及Th17相关细胞因子和转录因子；7. 我们利用CRISP/Cas9技术成功构建了miR-21敲除的小鼠模型。..结论：有关研究发现体外抑制miR-21对T细胞功能并无影响。而我们研究发现体内沉默miR-21抑制Th17细胞对中枢神经系统的浸润，明显抑制EAE发生发展，降低EAE小鼠小胶质细胞和巨噬细胞激活，有效降低CNS炎症浸润程度和脊髓脱髓鞘程度。体内和体外试验研究确定了miR-21调控EAE发生的靶向细胞是T细胞、小胶质细胞和巨噬细胞。miR-21可以通过调控T细胞、小胶质细胞和巨噬细胞来调控EAE的发生发展。确定了miR-21通过调控Rorγt信号通路调节Th17细胞，通过调节细胞自噬信号通路调控小胶质细胞和巨噬细胞，从而调控EAE的发生，为将来以此为靶点治疗EAE和MS提供了实验基础。