神经干细胞来源exosome抑制M1巨噬细胞极化促进脊髓损伤修复

Manipulation of macrophage polarization is becoming a hot spot in the field of spinal cord injury. Neural stem cell (NSC) could promote the functional recovery after spinal cord injury through suppressing the polarization of macrophage to M1 macrophage. Combined with the latest research and our pre-test results, NSC-derived exosome (NSC-Exo) may be very important for NSC to play an anti-inflammatory role, but its effects and mechanism of regulating macrophage polarization have not been reported. Therefore, in this study, we will construct bone marrow chimeric mice, establish spinal cord injury model, and then inject NSC-Exo into the caudal vein and local spinal cord. After treatment, MR-diffusion tensor imaging (MR-DTI) will be used to detect the axonal regeneration. The expression of M1/M2 macrophage and pro-inflammatory / anti-inflammatory cytokines in injured spinal cord and peripheral immune tissue, such as the spleen, will be tested by Real-time PCR and immunohistochemical staining. Furthermore, macrophage will be treated with NSC-Exo in vitro and then isolated to investigate the effect of NSC-Exo on the function and phenotype of macrophage, and meanwhile, the signal channels will be study, so as to determine whether NSC-Exo by acting on bone marrow-derived macrophages exert anti-inflammatory effects and promote the functional recovery of spinal cord injury, and it will provide new theoretical basis for the clinical application of NSC-Exo in repair of spinal cord injury.

调控巨噬细胞极化正成为脊髓损伤领域的研究热点。神经干细胞（NSC）可抑制巨噬细胞向有害的M1巨噬细胞极化从而促进脊髓损伤修复。结合最新研究成果和预实验结果，NSC来源exosome(NSC-Exo)是NSC发挥抗炎作用的关键，但其调控巨噬细胞极化的作用及机制还未见报道。因此，本研究拟在骨髓重建嵌合体小鼠基础上制作脊髓损伤模型，经静脉和脊髓局注射NSC-Exo治疗后，利用MR-DTI检测神经再生情况，利用Real-time PCR和免疫组织荧光染色检测脊髓损伤中心区和外周免疫组织中M1/M2巨噬细胞和促炎/抗炎因子表达情况，并在体外用NSC-Exo处理巨噬细胞后在细胞水平观察NSC-Exo对巨噬细胞功能和表型影响，并进一步阐述其信号通道，从而明确NSC-Exo是否通过作用于骨髓来源的巨噬细胞发挥抗炎作用并促进脊髓损伤功能恢复，为临床应用NSC-Exo修复脊髓损伤提供新的理论基础。

调控巨噬细胞介导的炎症反应正成为脊髓损伤领域的研究热点。近年来，神经干细胞(neural stem cell, NSC )的免疫调节越来越受到重视。我们前期研究首次证实神经干细胞及其上清液可抑制骨髓来源的巨噬细胞向有害的M1巨噬细胞极化从而促进脊髓损伤修复。但其机制还需要进一步研究。NSC来源的外泌体(NSC-Exo)能否作用于巨噬细胞并发挥抗炎作用还不清楚。因此，本研究拟建立脊髓损伤的模型并利用NSC-Exo治疗，然后观察小鼠的运动功能和轴突生长情况，以及损伤中心区巨噬细胞活化状态和促炎/抗炎因子表达情况，并在体外用NSC-Exo处理巨噬细胞后在细胞水平观察NSC-Exo对巨噬细胞功能和表型影响。本研究成功分离NSC-Exo。证实NSC-Exo具有抗炎和免疫调节作用，可抑制M1巨噬细胞的极化及其促炎性因子分泌。同时，NSC-Exo可通过抑制脊髓损伤巨噬细胞iNOS表达，减少脊髓组织的炎症反应并促进脊髓损伤后功能恢复。