MSCs外泌体介导的microRNA转运对癫痫海马小胶质细胞活化的作用机制研究

Microglia (MG) activation is an important pathological process in neurodegenerative diseases such as epilepsy. Our recent research indicated that mesenchymal stem cells (MSCs)-derived exosomes prevent inflammatory response and neural injury by altering microglia activity in the epileptic hippocampus (Long et al., PNAS, 2017), but the detailed effect and mechanism remains unclear. Activation of MG can be regulated by triggering receptor expressed on myeloid cells 2 (TREM2) signal (Yeh, Trends Mol Med, 2017) and microRNA is known to act as a key regulator of neuroinflammation as well (Henshall, Lancet Neurol, 2016). In a preliminary study we used the microarray technology to obtain a group of MSCs-derived exosomal miRNAs (miR-1268a, miR-1290, miR-199a-3p, miR-4299, let-7i-5p) thought to be involved in regulating MG activation and TREM2 signal. On the basis of these results we propose to conduct a series of in vitro (lipopolysaccharide-induced MG activation) and in vivo (Pilocarpine-induced epileptic model) experiments. Firstly, miRNA inhibition will be employed to select the MSCs-derived exosomal miRNA, which is responsible for regulating MG activation. After that, we will use a dual luciferase reporter gene assay to identify the target gene of selected miRNA associated with the TREM2 signal regulation (e.g. neutralization of sTREM2, preventing down-regulation of TREM2). Then, genetic engineering will be carried out to verify the regulatory role of MSCs-derived exosomal miRNA on MG activation and TREM2 signaling pathway (in vitro using lipopolysaccharide-induced TREM2-/- mouse derived MG activation, in vivo using Pilocarpine-induced TREM2-/- mouse model). Finally, we will elucidate the mechanism that MSCs-derived exosomes regulate microglia activation by the exosome-mediated miRNA transfer acting on TREM2 signal in the epileptic hippocampus. Understanding of this effect and mechanism may provide a theoretical basis of and experimental support for the treatment of neurodegenerative diseases including epilepsy by MSCs-derived exosomes.

小胶质细胞（MG）活化是癫痫等神经变性疾病的重要病理学特征。我们最近研究表明间充质干细胞（MSCs）外泌体可通过调节MG活化抑制癫痫海马炎症和神经损伤，但具体作用机制不清。鉴于MG活化主要依赖TREM2信号调节和miRNA是神经炎症的关键调节者，我们预实验利用microarray技术获得了一组由MSCs外泌体携带的可能调节MG活化及TREM2信号的miRNAs。本课题拟在此基础上，首先通过miRNA抑制筛选出外泌体调节体内外MG活化所转运的miRNA，随后依赖双荧光素酶报告基因检测验证这些miRNAs调节TREM2信号的作用靶点，进而利用基因修饰研究分析外泌体miRNA对MG活化及TREM2信号通路的调控作用，最终阐明MSCs外泌体通过其介导的miRNA转运作用于TREM2信号调控MG活化的作用机制。该作用机制的阐明将为MSCs外泌体治疗癫痫等神经变性疾病提供理论依据和实验支持。

小胶质细胞（MG）活化介导的神经炎症是癫痫（SE）等CNS疾病的发生发展的重要原因，前期研究表明间充质干细胞外泌体（MSC-EVs / MSC-Exo）对SE诱导小胶质细胞活化和神经损伤具有明显的保护作用，但MSC-EVs转运的具体活性物质及对小胶质细胞活化的调节机制不清。项目延续前期研究，通过LPS刺激小胶质细胞诱导活化后，应用其培养上清处理MSCs激发细胞的调节潜能，进而提取相应EVs（正常MSC-EVs设为对照），随后依赖microarray检测后获得与小胶质细胞活化调节相关的miRNAs（如miR199a-3p, miR-1290, miR-4299, miR-1268a, Let-7i-5p等）。在此基础上，结合TREM2在MG活化中的表达变化及调节意义，实验通过miRNA抑制剂测试发现，miR-199a-3p抑制后其对MG活化的失调节作用最为显著，证实MSC-EVs转运miR-199a-3p对MG活化的调节作用关键。同时针对MSC-EVs处理的活化MG行RNA-Seq检测，结合基因表达差异和蛋白互作网络分析，提示Fam53b、Mef2c、Sumo3、Fam49b、和Pds5a可能为miR-199a-3p作用于MG的靶点，因此实验通过双荧光素酶报告基因检测和免疫印迹实验分析发现Fam49b和 Sumo3为miR-199a-3p调节MG活化的靶基因。为进一步研究MSC-EVs转运miR-199a-3p对小胶质细胞活化的调节作用及机制，体外利用miR-199a-3p inhibitor和miR-199a-3p mimics处理活化的小胶质细胞，发现MSC-EVs调节小胶质细胞活化关键信号mTOR、p-mTOR、CD206、ARG1、TREM2和DAP12依赖miR-199a-3p转运。体内通过构建SE模型，诱导海马神经炎症及MG活化，随后接受miR-199a-3p mimics或miR-199a-3p-inhibited MSC-EVs处理，观察到MSC-EVs转运miR-199a-3p对海马炎症信号iNOS、TREM2、mTOR和Nrf2的调节作用不明显。综上提示MSC-EVs转运miR-199a-3p在体外对MG活化具有显著的调节能力，其在体内对海马炎症的调节影响尚待深入研究。