miR-133b修饰的MSCs来源外泌体对脊髓损伤后神经功能恢复的作用及机制研究

Unavailable of axonal regeneration is one of the leading causes of the poor functional recovery following spinal cord injury. Recent studies showed exosomes played an important role in delivery of miRNA. Additionally, experiments based on stem cell transplantation in cardiac injury revealed that beneficial effects of exosomes in tissue regeneration were similar to that of stem cells. Previously, we showed that miR-133b promoted neurite outgrowth and miR-133b-transfected MSCs effectively secreted exsomes enriched with miR-133b. Thus, we hypothesized that miR-133b exosomes derived from MSCs can promote axonal regeneration via activating pathways involved in axonal outgrowth by regulating target gene, finally, improve functional recovery after spinal cord injury. To verify the hypothesis, we investigate the effect of miR-133b exosomes on axonal regeneration and possible mechanism, as well as nerve regeneration and functional recovery following spinal cord injury by using primal neurons, rats spinal cord injury model, RNA interference, immunofluorescence, neural tract tracing and electrophysiology, via molecular, cellular, tissue and whole animal levels. The present project aims to combine the therapeutic effect and biological delivery effect for miRNA transfer of MSCs exosomes in spinal cord injury, which may provide a novel strategy for treatment of spinal cord injury.

神经传导束再生障碍是脊髓损伤后神经功能难以恢复的重要原因，迄今尚无有效的治疗方法。研究显示干细胞外泌体不但能够传递miRNA，而且对心脏等损伤组织的保护作用与干细胞类似。我们在前期研究中发现miR-133b能促进神经轴突生长，还证实基因修饰的骨髓间充质干细胞(MSCs)能够分泌富含miR-133b的外泌体。我们提出假说：MSCs来源的miR-133b外泌体，通过调控靶基因、激活轴突生长相关通路，促进轴突再生；并结合外泌体的治疗作用促进大鼠脊髓损伤后功能恢复。为了验证该假说，我们利用大鼠原代神经元和脊髓损伤模型，采用RNA干扰、免疫荧光、神经纤维示踪及电生理等技术，从分子、细胞、组织和动物整体水平多方面探讨miR-133b外泌体对神经轴突再生的影响及分子机制，以及对大鼠脊髓损伤后神经传导束再生及功能恢复的影响。本研究拟结合外泌体本身的治疗作用及miRNA传递作用，为治疗脊髓损伤带来新思路。

神经传导束再生障碍是脊髓损伤后神经功能难以恢复的重要原因，迄今尚无有效的治疗方法。本课题通过大鼠原代神经元和脊髓损伤模型，采用RNA干扰、免疫荧光、神经纤维示踪及电生理等技术，从分子、细胞、组织和动物整体水平多方面探讨miR-133b外泌体对神经轴突再生的影响及分子机制，以及对大鼠脊髓损伤后神经传导束再生及功能恢复的影响。首先建立大鼠脊髓损伤动物模型，发现损伤脊髓组织中miR-133b的表达量随着时间的推移显著下降。接着我们通过转染技术使得骨髓间充质干细胞（MSCs）过表达miR-133b，并分离、提取其中的外泌体，得到过表达miR-133b的外泌体。将miR-133b外泌体和miR-con外泌体分别移植至脊髓损伤大鼠中，发现miR-133b外泌体移植能够使得损伤脊髓组织中高表达miR-133b。通过BBB评分等测定大鼠运动功能恢复，结果提示miR-133b外泌体能够显著促进脊髓损伤后大鼠的运动功能恢复。另外我们通过免疫荧光、Western blot等方法发现miR-133b外泌体移植能够促进脊髓损伤后神经纤维及轴突的再生、减少脊髓组织中损伤腔隙的体积、增加神经元的存活等，并进一步揭示了miR-133b促进脊髓损伤后轴突再生的相关通路。本研究结合外泌体本身的治疗作用及miRNA传递作用，为脊髓损伤的治疗带来新策略。项目资助发表SCI论文2篇，待发表SCI论文1篇，培养硕士研究生1名，已毕业。项目投入经费18万元，支出13.5282万元，各项支出基本与预算相符。剩余经费4.4718万元，剩余经费计划用于本项目后续研究支出。