机械创伤诱导成纤维细胞向神经干/祖细胞重编程及其促进创面愈合的实验研究

Cutaneous innervation plays an important role in wound healing, cutaneous nerve repair is one of the key steps of wound healing, however, the underline mechanism is still unclear. According to previous studies, a large number of neural stem/progenitor cells could be induced in the wound tissues , which could promote nerve regeneration and wound healing. Nevertheless, their origins are not clearly clarified. Our previous work has shown that fibroblasts could be directly reprogrammed to neural stem/progenitor cells response to mechanical injury, which might be a new origin of neural stem/progenitor cells following wounding. Further study has preliminarily demonstrated that β-catenin pathway mediated the reprogramming of fibroblasts, and the inhibitors of differentiation (ID) were also significantly up-regulated following wounding, and there exists potential biding sites in promoter region of ID with the down stream proteins of β-catenin. Furthermore, ID proteins were showed to sustain stemess of neural stem cells by up-regulating of stemness genes and down-regulating differentiation genes. Thus we hypothesize that β-catenin pathway might regulate the expression of ID and mediate the reprogramming of fibroblasts to neural stem/progenitor cells following mechanical injuries. On the basis of above work, we aim to investigate mechanisms of β-catenin- ID pathway in the fibroblast reprogramming and wound healing at the molecular and cellular levels, as well as in vivo wound models combined with transgenic animals. This proposal would strengthen the understanding of the molecular basis and provide novel potential targets to wound healing therapy.

神经支配在创面愈合中发挥着重要作用，创面神经修复是创面愈合的重要环节之一，但其机制尚需阐明。研究表明创面局部存在大量神经干/祖细胞，其能明显促进神经修复和创面愈合，但其来源不明确。我们前期工作发现机械创伤可诱导成纤维细胞向神经干/祖细胞重编程，此途径可能是创面神经干/祖细胞的新来源。初步研究发现此过程受β-catenin信号途径调节；且分化抑制因子（ID）表达明显上调， ID转录区还存在β-catenin下游蛋白可能结合位点。既往研究表明ID能上调干性相关基因并抑制分化相关基因表达而增强神经干细胞的干性，因此我们推测机械创伤后β-catenin途径可能通过调节ID表达而介导成纤维细胞重编程。在此基础上，本项目拟通过体内外创伤模型结合转基因动物等途径，从分子、细胞、动物水平阐明β-catenin对ID的调控机制及其在机械创伤后成纤维细胞重编程及创面愈合中的作用，为促进创面愈合治疗提供新靶点。

神经支配在创面愈合中发挥着重要作用，创面神经修复是创面愈合的重要环节之一，但其机制尚需阐明。成纤维细胞是皮肤组织中主要的细胞类型，其在创面神经修复中的作用尚不完全清楚。既往的研究发现创伤后创面局部存在大量的类神经干/祖细胞，但其来源和在创面修复中的作用尚未阐明。因此我们推测机械创伤可能诱导成纤维细胞向神经干/组细胞重编程。本研究表明机械创伤后成纤维细胞并不能被重编程为神经干/祖细胞；但发现其具有类神经细胞样特性，在创伤组织中发现激活的成纤维细胞与神经轴突之间具有密切的连接；通过表达谱芯片分析发现成纤维细胞在创伤后被激活而高表达分化抑制因子（ID）主要为ID1和ID3, ID1/ID3能够增强成纤维细胞的自我更新和迁移能力，诱导成纤维细胞具有类星形胶质细胞样特性，全基因表达谱分析发现ID1和ID3在成纤维细胞中具有类似的调控功能，两者均能明显提高成纤维细胞促进神经轴突再生的能力，分析上调的差异蛋白发现ID1/ID3过表达的成纤维细胞中Itga6是表达最为丰富的基因，进一步通过与背根神经元共培养实验验证ID1/ID3能调控Itga6的表达而促进成纤维细胞引导神经轴突再生。移植ID1/ID3过表达的成纤维细胞到皮肤创伤组织或移植ID1/ID3过表达的慢病毒到耳朵创伤组织后发现两者均能提高创面的神经再生，并加快创伤愈合的速度，提高创伤愈合质量。此外移植ID1/ID3过表达的成纤维细胞还能明显促进坐骨神经损伤后的再生。本课题的研究发现成纤维细胞在创伤后能通过直接接触而促进局部神经再生，ID1/ID3-Itga6途径介导了这一过程。本项目的研究结果为后续周围神经损伤和局部神经病变导致的难愈性创伤愈合提供了潜在治疗策略。