组织特异性仿生微电流场构建及其对脊髓损伤后内源性神经干细胞的调控作用

The endogenous weak electic field plays an important role in the repair process after spinal cord injury. There is few studys on the properties of endogenous weak electic field in spinal cord injury and its association with the endogenous neural stem cells so far.Tissue-engineered flexible biomicroelectrodes designed in the last program supported by the National Natural Science Foundation are used in the repair of nerve injury successfully.Depend on the work of the last program and the anatomical structure,electrophysiological properties of the spinal cord, we intend to fabricate a specific biomimetic weak electric field to study its effection on endogenous neural stem cells in juryed spinal cord with new techniques of microelectronics,material science.The main content of this study including: development of novel biomicroelectrode core with the special anatomical structure of the spinal cord and well tissue compatibility,flexibility,electronic properties and less mechanical damage;analysis the weak electric field properties of the injuryed spinal cord and optimize the electric field parameters; observe behaviors of neural stem cells'activation and differentiation and migration in biomimetic weak field;confirm the related molecular mechanisms of the biomimetic electric field in regulating neural stem cells by inhibiting or over-expression the associated gene with the techniques of biotechnology.The tissue-specific biomimetic weak electric field fabricated in our study will provide a new approach on the reasearch of endogenous neural stem cells in injuryed spinal cord,also the repairation of spinal cord injury.

研究发现内生微电流场在脊髓损伤后修复过程中起重要作用。迄今对脊髓损伤后内生微电流场特点及其与内源性神经干细胞关系的研究较少。本研究团队在前一自然基金课题中设计出组织工程化柔性生物微电极并成功用于神经损伤修复。以此为基础，本课题应用微电子学、生物材料学等学科最新技术，结合脊髓的解剖结构和电生理学特征，拟构建脊髓组织特异性仿生微电流场用于研究其对脊髓损伤后内源性神经干细胞调控作用。本研究主要包括：研制生物相容性好、机械损伤小、导电性能稳定并适应脊髓特殊解剖学结构的微电极；分析损伤后脊髓内源性微电流场变化特点，优化仿生微电流场电生理参数；原位观察仿生电流场调控神经干细胞激活、分化、迁移的特点；利用生物技术抑制或过表达电流场相关基因，以明确电流场调控神经干细胞的相关分子机制。本研究所构建的脊髓特异性仿生微电流场，为脊髓内源神经干细胞调控研究提供了新的途径，进一步为脊髓损伤的修复研究提供新的策略。

本课题根据前期在生物性神经微电极修复神经损伤研究方面取得的经验，模拟体内的内生电场特点，在体外构建了仿生弱电流场细胞培养体系，通过无血清悬浮培养方法，成功培养出多分化潜能旺盛的脊髓源性神经干细胞。弱电流场实验结果表明，条件参数设定为电场60mv/mm,液面高度为800um时为适宜电场微环境参数，仿生弱电流场能够促进脊髓源神经干细胞的增殖，并且促进神经干细胞向神经元分化。.进而，根据SD大鼠的脊髓参数，制作脊髓损伤刺激电极。将SD大鼠进行脊髓损伤造模后给予微电流刺激，并通过功能行为学BBB评分、Western-Blot检测、PCR检测、免疫荧光技术、激光共聚焦技术及全息合成技术评估，同时，随着微电流刺激作用时间的延长，脊髓损伤SD大鼠的BBB评分逐渐改善；Western-blot、RT-PCR及免疫组化的结果均提示在脊髓灰质内存在一定量的内源性神经干细胞；脊髓损伤后内源性神经干细胞多向星形胶质细胞分化，极少向神经元分化；脊髓电刺激可在一定程度上激活脊髓内源性神经干细胞，并通过抑制Notch信号通路以促进内源性神经干细胞向神经元分化，而抑制其向星形胶质细胞分化，促进损伤脊髓的自我修复。.研究发现脊髓损伤发生后主要存在急性损伤期和继发性反应期；在急性期，有大量的M2型巨噬细胞渗入；在脊髓损伤持续期，经典的M1型巨噬细胞被大量激活，渗入脊髓损伤局部区域，通过将脊髓损伤区域巨噬细胞的IRF5敲除可促进巨噬细胞由M1型向M2型转变，可减轻退髓鞘变化、神经纤维丢失、运动功能明显恢复。.本研究为应用脊髓神经电刺激干预脊髓损伤后修复过程及其相关机制提供依据，同时IRF5可能为治疗脊髓损伤的一个新靶点。