抑制RhoA表达与活化对周围神经缺损后再生轴突髓鞘化的作用及机制研究

Remyelination is an essential process for axonal regeneration but it did not be emphasized in previous published works on the bioengineering repair for the peripheral nerve defect (PND). It had been identified that suppressing RhoA signal pathway can improve the axonal outgrowth. While up to date, the relationship of RhoA and remyelination in peripheral nerve defect is not clear yet. Our preliminary data showed RhoA is significantly upregulated in Schwann cells (SCs) after peripheral nerve injury, which indicated RhoA plays important roles in demyelination and remyelination after PND. Previously, we had utilized self assambling peptide nanofiber scaffold (SAPNS) combined with RhoA siRNA or inhibitor to treat the spinal cord injury and PND, we also developed a noval artificial nerve conduit with PLGA/SAPNS/SCs. In present project, we aimed to detect the effects of the noval conduit for reconstructing the defected sciatic nerve, to explore whether suppressing RhoA pathway can facilitate remyelination during the bioengineering of PND. We also plan to knockdown/ upregulate the RhoA expression or inhibite RhoA activation in vitro and in vivo to explore the mechanism of RhoA in different steps of remyelination which include SCs proliferation, survival, migration, initiation of myelination, mature of myelin and the expression the important functional proteins of myelin. The overall aim of this project is to search a new therapeutic strategy for PND by targetting the RhoA pathway to facilatate the remyelination and axonal regeneration.

周围神经缺损（PND）后的再生轴突只有被髓鞘化才具备正常传导功能，而目前PND修复研究对髓鞘化却重视不够。抑制RhoA信号通路已被证实能促进轴突再生，但它与再髓鞘化的关系尚不明确。我们最近发现PND后施万细胞(SCs)内 RhoA表达大幅上调，提示RhoA在脱髓鞘及髓鞘化中发挥重要作用。本组近来分别将自聚合肽纳米纤维材料（SAPNS）结合RhoA的siRNA和抑制剂用于修复脊髓损伤和PND，并构建了PLGA/SAPNS/SCs复合人工神经桥接材料。在上述基础上，本研究拟探明：PLGA/SAPNS/SCs修复PND过程中抑制RhoA信号通路可否在促进轴突再生的同时增强髓鞘化；拟通过降低或增高表达、抑制活化等手段阐明RhoA对SCs增殖、存活、迁移、髓鞘形成及髓鞘重要功能蛋白表达的影响，揭示RhoA参与再髓鞘化各环节中的作用机制。为今后以RhoA为靶点促进髓鞘化，提高PND治疗效果提供新思路。

项目背景：周围神经缺损（peripheral nerve defect, PND）是临床常见疾患，PND不仅影响患者的生活质量，还对家属和社会带来沉重的负担。我们通过文献分析及前期工作基础和预实验结果等综合分析提出科学假设：RhoA参与PND后再生轴突髓鞘化的过程，抑制RhoA信号通路可能同时促进轴突再生和髓鞘化。本课题主要是验证上述假设并希望能为今后以RhoA为靶点促进轴突再生及其髓鞘化，为提高PND治疗效果提供新思路。.主要研究内容：构建一种新型的复合人工神经桥接材料；构建RhoA-shRNA及RhoA过表达慢病毒；在人工神经中添加病毒载体及RhoA抑制剂，然后用于桥接缺损的神经；研究RhoA低表达、高表达及活化抑制对施万细胞增殖、存活及迁移的影响；RhoA对髓鞘重要功能蛋白表达的影响；研究RhoA影响髓鞘形成的机制。.重要结果：建立了一种新型施万细胞（SCs）分离纯化的方法；建立了从外周血获取间充质干细胞（PBMSCs），经体外大量增殖后将其诱导分化为SC的方法；构建3种含细胞或不含细胞的复合人工神经桥接材料；探明了RhoA在周围神经发育和损伤后的表达时空变化；探明了RhoA对施万细胞增殖、存活及迁移的影响；发现了RhoA信号通路对施万细胞的分化及去分化均有重要的作用；首次发现RhoA可以调控微管的稳定性从而引起一系列生物学功能的变化。.主要成果：获得国家发明专利授权1项， 发表各类论文12篇， 参加学术会议26人次；培养博士生6人,硕士生10人。