壳聚糖神经移植物体内降解物壳寡糖对神经轴突生长的影响及机制研究

The artificial nerve grafts have been effectively bridged peripheral nerve defects with satisfactory functional reconstruction. Numerous studies showed that, the chitosan constructed nerve grafts not only provide a good channel for nerve regeneration; its degradation products, chitooligosaccharides (COS), can also promote nerve regeneration. This is mainly due to the promotion effects of COS on growth of neurons and proliferation of Schwann cells (SCs). Our previous study has shown that COS accelerate cell cycle by miR-27a/FOXO1 axis, lead to a stimulation of SC proliferation, and thus eventually facilitate nerve regeneration. Till to date, however, the potential roles and the involved molecular mechanisms of COS on axon growth remain unclear. The current project towards these issues and it will be carried out as described below. Firstly, we will separate chito-oligomers with high purity single degrees of polymerization (DPs) from a mixture of COS by using hydrophilic interaction liquid chromatography (HILIC), and the weight of chitooligosaccharide is hundreds mg. Secondly, we are going to systematically investigate the effects of COS monomer at different concentrations on nerve axon growth by using neuronal cell body - axon isolation cultivation model, then get the proper COS monomer and its optimal concentration for axon growth. Thirdly, bioinformatics analysis will be used to identify the involved molecular signaling pathways and key regulatory factors of differentially expressed proteins of axons and differentially expressed genes. Taken together, our study will study the effect of COS-mediated signaling cascades on axon growth, thus to reveal the characteristics and mechanisms of the degradation products of chitosan-COS on promoting axon growth. Our data will provide a theoretical basis for the clinical application of chitosan constructed nerve grafts.

人工神经移植物是临床治疗周围神经缺损的有效措施,我们研究发现壳聚糖神经移植物在修复神经缺损过程中不仅为神经再生提供通路，还能促神经再生,主要是壳聚糖体内降解产物壳寡糖能够促进施万细胞增殖和神经元生长,同时还发现壳寡糖通过miR-27a/FOXO1调节细胞周期促进施万细胞增殖来促进神经再生，但壳寡糖促进神经元轴突生长的规律及机理未见报道。我们拟通过已建立的亲水相互作用色谱将壳寡糖混合物分离成9个高纯度壳寡糖单体（壳二糖到壳十糖）；建立神经元胞体-轴突隔离培养模型，研究各壳寡糖单体对神经轴突生长的影响，筛选出促进作用最好的壳寡糖单体及最佳浓度；采用生物信息学分析轴突差异表达蛋白和神经元胞体内差异基因之间的信号通路以及核心调控因子，研究壳寡糖介导的信号回溯对轴突生长的影响；该项目将揭示壳聚糖神经移植物降解产物壳寡糖促进轴突再生的规律及其作用机制，为壳聚糖神经移植物和壳寡糖的应用提供理论支撑。

人工神经移植物是临床治疗周围神经缺损的有效措施,我们研究发现壳聚糖神经移植物在修复神经缺损过程中不仅为神经再生提供通路，还能促神经再生,但壳寡糖促进神经元轴突生长的规律及机理未见报道。本项目按照项目申报书研究内容开展研究，通过已建立的亲水相互作用色谱将壳寡糖混合物分离；建立神经元培养模型和神经损伤模型来研究壳寡糖对神经轴突生长的影响，确立壳寡糖的效应；采用生物信息学分析差异表达蛋白和信号通路以及核心调控因子，揭示壳寡糖介导的效应对外周神经细胞间的相互作用、轴突生长及再生微环境的影响。该项目的开展，揭示壳聚糖神经移植物降解产物壳寡糖促进轴突再生的规律及其作用机制，为壳聚糖神经移植物和壳寡糖的应用提供理论支撑。研究超额完成了项目既定目标，共发表标注项目号的SCI论文15篇，获得发明专利1项，申请发明专利5项，并指导6名参与该项目的硕士研究生及1名博士研究生顺利毕业。