嗅鞘细胞以巨噬细胞为靶点促进脊髓损伤修复的抗炎机制研究

The anti-inflammatory effect of olfactory ensheathing cells (OECs) began to concern recently. But whether grafted OECs could promote axonal tisuue regeneration and repair after spinal cord injury by manipulating bone marrow-derived macrophage to regulate inflammation response remain unclear. Therefore, in this study, we will reconstruct bone marrow chimeric mice, establish the model of spinal cord injury and then eliminate bone marrow-derived macrophage within damage area by using the conditional macrophages strategy. After OECs with a green fluorescent protein (GFP-OECs) were transplanted into spinal cord, the movement function of mice and axon growth were observe, as well as inflammatory cytokines and macrophages within the epicenter of injured spinal cord. In addition, OECs are incubated with macrophage in vitro to detect the effect of OECs on the macrophage phagocytosis and polarization and to investigate the relation between anti-inflammatory effect of transplanted OECs via regulating macrophage and the functional recovery of spinal cord injury. The anti-inflammatory effect of OECs against SCI via regulating macrophage polarization may be another important mechanism responsible for functional improvement. It will provide a new theoretical basis for OECs clinical applications to repair spinal cord injury.

近年来，嗅鞘细胞的抗炎作用开始受到关注，但以巨噬细胞为作用靶点的抗炎机制在OECs移植促进轴突再生及修复脊髓损伤的作用还未明确。因此，本课题拟在骨髓重建嵌合体小鼠的基础上建立脊髓损伤模型，并利用条件性巨噬细胞消融策略消除损伤区骨髓来源的巨噬细胞，然后将携带绿色荧光蛋白的OECs（GFP-OECs）移植入体内，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，并且将OECs与巨噬细胞共培养后在细胞水平观察OECs对巨噬细胞吞噬等功能和极化影响，探讨OECs是否通过作用于损伤区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OECs修复脊髓损伤提供新的理论基础。

本项目着眼于脊髓损伤（spinal cord injury, SCI）修复细胞移植治疗中疗效最确切的嗅鞘细胞为研究对象，着眼于嗅鞘细胞（olfactory ensheathing cell, OECs）移植对脊髓损伤中心巨噬细胞表型、炎性因子等功能以及巨噬细胞极化相关信号通路的影响，证明了嗅鞘细胞通过调节巨噬细胞极化发挥抗炎作用及其在脊髓损伤修复中的意义。此外，目前尚无文献报道OECs移植后对SCI的基因调控作用，特别是miRNAs介导的转录后基因调控，本研究首次阐明OECs移植能够逆转SCI后异常表达的miR-199a-5p，为研究miRNAs介导的转录后基因调控作用提供了新的思路。本研究首次揭露了OECs对Slit/Robo信号通路的基因调控作用，揭示了OECs移植治疗SCI的又一个新机制：OECs移植诱导大鼠下调miR-199a-5p的表达，下调的miR-199a-5p减弱了对Slit/Robo信号通路中轴突导向分子Slit1、Robo2和srGAP2的转录后基因沉默效应，增加了Slit1、Robo2和srGAP2的蛋白水平，从而促进SCI的修复，此发现为更全面地理解OECs移植修复SCI的机制提供了新见解和理论支撑。我们将基因共表达网络分析、系统聚类和差异表达基因相互比较等方法结合应用起来，首次描绘出脊髓损伤后成年小胶质细胞的时间转录组图及共有的核心基因程序。我们重点提出损伤激活的小胶质细胞（injury-activated microglia, IAM）具有核心基因表达程序，包括：免疫反应、清除（脂质代谢和吞噬）和组织修复（基质重构和分泌因子）。我们也描述了脊髓损伤后小胶质细胞不同转录功能、分阶段的激活作用，包括其早期阶段的细胞周期和运动的渐进调节，中间阶段的轴突趋化作用和离子通道活动，后期阶段的基质调节作用。值得注意的是，IAM表现出显著的抗炎基因的富集强化，为小胶质细胞在脊髓损伤中的促修复作用提供了强有力的支持。