负载活性纳米水凝胶的双仿生组织工程神经递送多信号协同调控炎症及髓鞘化促进周围神经再生的研究

Peripheral nerve injury remains a major challenge in clinic. Tissue engineering serves as a promising strategy for treatment of this injury. The main topic for tissue engineering is to re-construct suitable microenvironment for guiding regenerative axons into the distal nerve stump, meanwhile, regulating the surrounding inflammatory factors, thus facilitating myelinated nerve regeneration. Our previously works have developed aligned acellular cauda equine by using chemical decellularizition method. Based on above, the current project is aimed to construct a composite artificial nerve graft by combing acellular cauda equine and self-assembling nanofiber hydrogels RAD/10Panx and RAD/IKVAV-RGI that mimicking the natural nerve tissue in both composition and microstructure for 15 mm long-distance defects repair in rat sciatic nerves. The treatment efficiency will be comprehensively evaluated regarding structural and functional recoveries. Moreover, the precise mechanisms underlying how the peptides RAD/10Panx and RAD/IKVAV-RGI function as biological cues and regulate early inflammatory response as well as promote myelination of regenerated axon will be thoroughly deciphered. We believe our project will will provide a novel strategy and a solid theoretical foundation for long-distance peripheral nerve defects treatment.

周围神经损伤修复是亟待解决的临床难题。构建适宜的神经再生微环境，通过引导近端神经纤维有序生长以提高神经再生速度，同时调控局部炎症及再生神经髓鞘化以提高神经再生质量，从而促进周围神经再生是当前组织工程神经研究的重要方向。本项目拟在申请人前期研究的基础上，以细胞凋亡辅助去细胞法制备结构和成分双仿生的马尾神经ECM作为支架材料，复合两种功能性自组装多肽RAD/10Panx与RAD/IKVAV-RGI所形成的纳米纤维水凝胶，构建组织工程神经，用于修复大鼠坐骨神经15mm长节段缺损，观察其修复效果，并从两种功能性多肽片段10Panx与IKVAV-RGI递送多个生物信号协同调控早期炎症及再生轴突髓鞘化的角度，探讨其作用机制，为周围神经长节段缺损的修复提供新的治疗策略。

周围神经损伤修复是亟待解决的临床难题，而研发能够有效替代自体神经的移植替代物是目前国内外研究的热点。构建适宜的神经再生微环境，通过引导近端神经纤维有序生长以提高神经再生速度，同时调控局部炎症及再生神经髓鞘化以提高神经再生质量，从而促进周围神经再生是当前组织工程神经研究的重要方向。基于此，本项目首先建立了细胞凋亡辅助去细胞法制备神经源性ECM的标准化方案，发现5 μM喜树碱处理马尾神经24小时即可获得最佳去细胞效果，并在保留神经ECM结构和生化成分方面较传统十二烷基硫酸钠（SDS）去细胞法更具优势。团队设计并制备了源自血管内皮生长因子（VEGF）的多肽KLT，通过体内实验证明含有KLT的水凝胶能够促进周围神经缺损移植段的血管化，并能够与源自层粘连蛋白（Laminin）的多肽IKVAV协同促进损伤周围神经的轴突再生和髓鞘化。随后团队又将IKVAV与脑源性神经营养因子（BDNF）来源的多肽RGI结合，制备双功能化自组装多肽水凝胶，体内外实验证实二者的协同作用可以明显改善轴突再生，促进再生神经的再髓鞘化和运动功能恢复。在此基础上团队将RGI添加到IKVAV短肽序列中，形成兼具BDNF和Laminin生物活性的功能性多肽片段IKVAV-RGI，并将其与具有抗炎、抗凋亡功能的10Panx功能短肽接合到自组装多肽RADA16-I的C端，形成兼具抗炎及修复功能的水凝胶RAD/IKVAV-RGI/10Panx，该水凝胶可在促进再生神经纤维的生长、SCs分泌神经营养因子及髓鞘化的同时，调控巨噬细胞向M2型极化，从而兼具抗炎活性。将该水凝胶吸纳入冻干的马尾神经ECM，制备成为负载活性纳米水凝胶的双仿生组织工程神经，桥接大鼠坐骨神经缺损实验证实其能够显著改善损伤坐骨神经的电信号传导功能，并在损伤早期有效维持支配肌肉的活性、明显改善损伤后的神经-肌肉再支配，从而高效恢复患肢功能。