周围神经ECM通过NCAM途径促进再生轴突集束的机制研究

Nerve conduits transplantation is an important means to repair peripheral nerve defects, However, the disorganized and disorderly regeneration of intraductal axons hinder the function recovery. Axonal fasciculation plays an important role in the orderly and directionally growth of nerve fiber during embryonic development, which is mediated by nerve cell adhesion molecule (NCAM). but the distribution mechanism of NCAM in axons of peripheral nerve and its effect on axonal fasciculation are remain unclear. our in vivo and in vitro experiments showed that the ECM of peripheral nerves can specifically cause axonal fasciculation, accompanying with the increased NCAM expression in axon. thus, we propose a hypothesis that The ECM proteins of peripheral nerve can bind with NCAM molecule on the surface of axolemma, Increase the concentration of NCAM in the axon region, thereby promoting axonal fasciculation. In this study, we'll identify the ECM protein binding to NCAM by GST pulldown and Co-IP; By increasing or suppressing the function of this ECM protein we can demonstrate its role in regulating NCAM's location on axolemma, together with axonal fasciculation assay we will clarify the mechanism that peripheral nerve ECM proteins regulate axonal fasciculation by increasing NCAM enrichment in axons. This study is expected to provide a theoretical basis for the raise of new nerve repair strategy from the aspect of axonal orderliness.

神经导管移植是修复周围神经缺损的重要手段，但导管内轴突的散乱无序再生阻碍了神经功能恢复。由神经细胞粘附分子（NCAM）介导的轴突集束被证明是维持胚胎发育过程中轴突生长的有序性和方向性的必要条件。然而NCAM在周围神经轴突的分布调控机制及其对轴突集束的作用尚不清楚。我们通过在体及离体实验发现周围神经ECM能特异性引起轴突集束，同时伴随着轴突NCAM增加，推测周围神经ECM蛋白能与轴膜表面的NCAM分子结合，吸引其在轴膜的定位，增加轴突区域NCAM的富集度从而促进轴突集束。本课题首先通过GST pulldown、免疫共沉淀鉴定出与NCAM结合的ECM蛋白；其次，增加和抑制此ECM蛋白的功能证明其调控NCAM在轴突定位的作用，并结合轴突集束程度确定：周围神经ECM蛋白通过增加NCAM在轴突的富集度而促进轴突集束。此研究有望从轴突再生有序性方面为神经修复新策略的提出提供理论依据。

神经纤维的随机无序再生是阻碍神经损伤后功能恢复的重要因素。基于引导信号的促神经有序再生策略在实际应用中易受体内复杂微环境因素的干扰，且对制备技术要求较高，导致大多数现有促神经有序再生研究的转化应用难度较大。. 本项目利用组织脱细胞和基质凝胶化技术构建了多种具有组织特异性的基质水凝胶。结合神经组织3D培养我们发现：外周神经细胞外基质能够在不依赖引导信号的条件下引发有序神经束结构的形成。. 对其作用机制的进一步研究证明：外周神经细胞外基质成分中的六型胶原（COL6）能够与神经细胞黏附分子1（NCAM1）的胞外结构域结合，提高后者在轴膜的稳定性并藉由NCAM1的嗜同性结合作用促进有序神经束结构的形成。此研究揭示了一种尚未被充分探索的神经束形态发生范式（即通过匀质的ECM成分变化指导散乱神经纤维向平行神经束的发展）。此过程完全由神经纤维的自组织驱动，因此克服了传统促神经有序化策略对工艺技术要求高，且易受体内复杂微环境因素干扰的问题。.基于大鼠坐骨神经长段缺损的动物实验表明：负载COL6的匀质水凝胶能够显著提高再生神经的有序性和投射准确率。大鼠的神经功能水平较对照组有着明显恢复。. 考虑到COL6由于分子量巨大而存在制备困难且免疫原性较强的问题。本项目发表的第二篇论文发现分子量较小的COL6A2亚基具备独立的促神经自组织有序化作用。与完整COL6相比，COL6A2表现出更低的免疫原性和重组制备的可行性，在转化应用方面较COL6更具优势。