基于骨髓神经嵴细胞的组织工程神经修复周围神经缺损及其机制研究

Nerve transplantation to bridging peripheral nerve defect is the effective measurement in clinic therapy. Compared with autograft nerve transplantation, the transplantation of material tube shows less effectiveness in rescuing long-distance or thick nerve defect. Our previous work indicated that artificial nerve transplantation with stem cells could markedly improve the repair effect. To investigate the repair effect of tissue engineering nerve based on bone marrow neural crest cells (BM-NCCs), methods such as function tests, electrophysiological detection, axon fluorescence tracking, and morphological observation would be performed to evaluate the efficacy on bridging nerve defect. To illustrate the profiles and dynamic tendency of the gene and protein expression involved in various biological processes in the regenerating micro environment, including cell survival, inflammation reaction, angiogenesis, cell proliferation, cell migration, axon regrowth, remyelination etc., the biological information from the gene transcriptome and protein expression of the regenerated nerve tissue would be systemically analyzed based on the high-throughput detection. Extracellular vesicles （EVs） derived from BM-NCCs would be isolated, and applied to observe the repairing or regulating effect to neuron, vein endothelial cells, Schwann cells etc.. To screen and validate the biological activated molecules related with nerve repair, the biological information of RNA and protein contained in EVs derived from bone marrow neural crest cells would be systemically analyzed based on the high-throughput detection. This study will reveal the micro environment improvement action mechanism of BM-NCCs via secreting EVs to repair nerve defect, and will provide new idea for enhancing the repair efficacy of stem cells on peripheral nerve defect.

神经移植物桥接术是临床治疗周围神经缺损的有效措施，单独由材料构建的移植物修复长距离或粗大的神经缺损时，其修复效果与自体神经相比还有较大差距。我们前期研究表明含干细胞的神经移植物能显著提高其修复效果。本研究拟构建基于骨髓神经嵴细胞（BM-NCCs）的组织工程神经，采用行为学、电生理学、荧光示踪、形态学等方法观察BM-NCCs对神经缺损的修复作用；采用高通量技术动态检测分析神经再生组织的基因转录组和蛋白，明确微环境中血管新生、轴突再生、髓鞘再形成等过程的基因-蛋白表达模式。分离BM-NCCs分泌的囊泡（EVs），体外观察EVs对神经元、施旺细胞、血管内皮细胞等的修复或调控作用；采用高通量技术检测EVs所含非编码RNA和蛋白，筛选并验证与神经修复相关的生物活性因子。本研究可初步揭示BM-NCCs通过分泌EVs改善再生微环境修复神经缺损的作用及机制，为提高干细胞修复周围神经缺损的效果提供新的思路。

周围神经损伤后的修复与再生是神经科学基础与临床研究者的重要目标，以减轻患者痛苦和社会及家庭经济负担。本研究通过构建基于神经嵴细胞（NCCs）的组织工程神经，探究其促进动物神经缺损后修复与再生的作用与机制。大鼠模型的行为学、电生理检测、荧光示踪、形态学结果都表明，NCCs或NCCs分泌的囊泡联合应用生物材料导管/支架可有效修复神经损伤，促进缺损神经再生。通过对损伤后修复过程中神经组织生物学信息变化的高通量检测与分析，明确再生微环境中细胞存活、血管新生、细胞增殖、细胞迁移、轴突再生、髓鞘再形成等生物学过程均受NCCs及分泌的胞外囊泡所转运物质的良好调控。通过对NCCs分泌体以及胞外囊泡内容物进行检测与分析，并验证囊泡所传递的miRNA等活性因子在再生微环境中起到关键的生物学过程调控作用，揭示利于启动神经修复通路的相关信号通路和靶点，不仅可修复感觉和运动神经元本身，还可通过调控微环境中的施旺细胞和血管内皮细胞等其他细胞的生物学功能加强其促神经再生作用。总而言之，本研究工作通过对NCCs旁分泌作用与机制的探究，揭示其改善微环境修复神经损伤和实现周围神经功能重建的良好效应，对推动将来NCCs组织工程神经的转化应用，以及NCCs及胞外囊泡传递的促再生信号与物质的开发利用，提出更好的综合治疗策略提供了重要的理论和实验依据。