IRF5-siRNA介导的M2型小胶质细胞外泌体激活内源性神经干细胞治疗脑梗死的机制研究

Modern medicine has no effective measure for the neural function recovery in cerebral infarction (CI). To mobilize the proliferation, directional migration and differentiation of endogenous neural stem cells (eNSCs) to repair damaged neurons is expected to fundamentally solve the problem. Recent studies found that, by altering the microenvironment of the damaged brain, M2-type microglia could activate the proliferation of eNSCs and induce directional migration and differentiation. Down-regulating the expression of IRF5 in microglia can promote the transformation of microglia from M1 to M2. In our previous study, M2-type microglial cells were co-cultured with the NSC isolated from EGFP-Nestin transgenic mice, and the proliferation and differentiation of NSC were observed in vitro. Meanwhile, The MCAO model was constructed with Nestin-Cre/ERT2 transgenic mice, and the IRF5-siRNA intravenously carried by lipid nanoparticles was injected into MCAO mice to observe the proliferation, migration and differentiation of eNSC in vivo. The components in exosomes were analyzed in order to find the possible target protein or target nucleotide fragments in the mobilization of eNSC. By changing the brain microenvironment, the eNSC was activated to directional migrate and proliferate, differentiate, then the brain damage caused by CI was repaired. This study provides a new idea of the treatment of CI.

现代医学对脑梗死后神经功能的修复没有有效的对策。动员内源性神经干细胞(eNSC)增殖并定向迁移和分化来修复受损神经元有望从根本上解决问题。近年来研究发现M2型小胶质细胞可通过改变损伤脑内的微环境来激活eNSC的增殖并发生定向迁移和分化。特异性下调小胶质细胞内IRF5的表达，可促进小胶质细胞由M1型转化为M2型。本研究在体外细胞水平上，将脂质体包裹的IRF5-siRNA诱导的M2型小胶质细胞与EGFP-Nestin转基因小鼠的原代NSC共培养，观察NSC的增殖、分化情况；同时，用Nestin-Cre/ERT2转基因小鼠构建MCAO模型，并将IRF5-siRNA静脉注射入MCAO小鼠中，观察eNSC在体的增殖，迁移和分化状况;分析外泌体的成分，以期寻找动员eNSC可能的靶蛋白或靶核苷酸片段。本研究通过改变脑内微环境，激活eNSC增殖、定向迁移和分化修复脑损伤，为脑梗死治疗提供新思路。

本项目主要研究IRF5-siRNA介导的M2型小胶质细胞外泌体治疗脑梗死的机制。通过IRF5-siRNA对M1型小胶质细胞中IRF5的敲降，改变小胶质细胞的表型，抑制缺血后脑组织炎性反应。体外细胞实验：利用白介素等因子对BV2细胞进行诱导分化为M1、M2型小胶质细胞，PCR实验证明细胞分化成功，western blot 显示M1型小胶质细胞中IRF5的表达水平远高于M2。体内实验：为了量化小胶质细胞的激活，使用小胶质细胞特异性标记 CD11b和 IBA1进行计数，ImageJ 分析小胶质细胞阳性率大于 99%；采用转棒试验对实验对象进行了运动能力评价；采用免疫荧光技术，对实验动物海马及皮层区小胶质细胞原位染色，确定小胶质细胞在不同脑区的激活状态。治疗效果：在MACO小鼠模型上，经鼻注射hADSC治疗方式，可改善脑卒中模型鼠的神经功能评分、运动平衡功能及小胶质细胞的静息激活状态；经脂肪干细胞来源外泌体治疗方式，可以改善中风小鼠肢体协调能力的损伤。本项目的结论是，脂肪干细胞外泌体可明显改善神经功能损伤，且随着时间增加，神经功能恢复越加明显。本研究为脂肪干细胞外泌体治疗脑卒中患者预后身体协调功能提供新的思路。.