低温对脑外伤后小胶质细胞外泌体释放、神经前体细胞增殖分化与迁移作用及关键机制

Mild hypothermia has been recognized to be an effective measures for cerebral protection, and has an effect on the improvement of neurological dysfunction after traumatic brain injury (TBI). However, the effects and molecular mechanisms of hypothermia on neural progenitor cells proliferation, differentiation and migration after TBI have not been elucidated. The aims of this item are to explore the secretion of microglia exosomes and its effect on neural progenitor cells proliferation, differentiation and migration after TBI in the model of cell strain-induced injury, fluid percussion brain injury in rats, controlled cortical injury in mice and microglial depletion mouse model. The proteome profiler mouse cytokine array kit is used to assess the expressions of cytokine proteins in microglia exosomes, and specific cytokine inhibitors are used to inhibit the secretion of specific cytokines in microglia exosomes so as to discover the cytokines that may associated with the proliferation, differentiation and migration of neural progenitor cells after TBI. Finally, the technologies of virus transfection and specific inhibitors are applied to regulate the expression of specific cytokines and its downstream signaling molecules so as to clarify the molecular mechanisms of hypothermia on neural progenitor cells proliferation, differentiation and migration after TBI. It is hopefully explored that the role of hypothermia on neural repair and regeneration after TBI and possible mechanisms of cellular signal transmission, which may find a new therapy target for neural repair and regeneration after TBI.

低温已经成为颅脑创伤后脑保护技术的重要手段之一，可显著改善颅脑外伤患者神经功能障碍。有关低温在颅脑创伤后对神经前体细胞增殖分化及迁移的作用及其机制尚不明确。本项目通过建立细胞体外机械损伤模型，小鼠液压脑损伤模型，小鼠控制性脑皮质损伤模型及自体小胶质细胞活化抑制小鼠模型，探究低温对颅脑外伤后小胶质细胞外泌体分泌的影响，及这种影响对颅脑外伤后神经前体细胞增殖分化及迁移的作用。在此基础上，运用细胞因子蛋白鉴定试剂盒分析低温后小胶质细胞外泌体细胞因子成分及含量的改变，并通过特异性细胞因子抑制剂抑制小胶质细胞特定细胞因子的外泌体释放来明确与神经前体细胞增殖分化及迁移相关的细胞因子及其具体作用。最后，通过使用与神经前体细胞增殖分化及迁移相关的下游信号通路的特异性抑制剂及慢病毒介导的关键基因敲低技术来揭示相关细胞因子的下游调控信号通路。从而阐明低温在颅脑创伤后对神经前体细胞增殖分化及迁移的作用及其机制。

我们通过建立小胶质细胞“炎症反应损伤低温模型”，提取小胶质细胞来源外泌体处理神经前体细胞。我们发现神经前体细胞可以摄取小胶质细胞来源外泌体，并且发现损伤炎症反应后33℃低温处理后的小胶质细胞所分泌的外泌体能促进神经再生：促进神经前体细胞的增殖，并且能促进神经前体细胞向神经元分化。进一步在建立小鼠“控制性皮质损伤模型”的基础上，我们发现静脉注射小胶质细胞来源外泌体后，炎症反应后33℃低温处理后的小胶质细胞所分泌的外泌体能促进小鼠脑损伤后运动功能及任职功能的恢复。损伤炎症反应后33℃低温处理后的小胶质细胞所分泌的外泌体IL-1ra，CCL5 释放水平上升，TNF-α及CXCL10释放水平下降，可能作为调控神经前体细胞生长因子的途径影响神经前体细胞的增殖分化。我们的研究阐明低温在颅脑创伤后对神经前体细胞增殖分化的作用，进一步阐明颅脑创伤早期低温治疗与颅脑创伤神经修复再生的相互关系，为颅脑创伤早期脑神经修复再生提供新途径。