MicroRNA-17-92 cluster 缓释微球组织工程化神经移植物的构建及其应用于修复大鼠周围神经缺损的研究

Tissue engineered nerve grafts (TENGs) represent a promising alternative to autologous nerve grafts used for peripheral nerve repair, and have become a focus in research of peripheral nerve regeneration. In the RNA-seq study with 0.5cm long nerve tissues at different time points from both the proximal and distal nerve ends in the rat sciatic nerve transection model. A set of genes during the process of nerve injury were identified. Most time-dependent differentially expressed genes were functionally classified into different categories. Integrated with the mRNA and miRNA expression profiles, miRNA target gene pathway enrichment analysis, we developed the mRNA-miRNA interaction network to reveal the post-transcriptional landscape of peripheral nerve injury. The results of the bioinformatics analysis indicated that miR-17-92 cluster not only plays a crucial role in neural development, but also the key gene in peripheral neural regeneration. Based on this premise, this project aims to construct a novel TENG that is featured by a controlled release PLGA microsphere drug delivery system through immobilizing a miR-17-92 cluster overexpression plasmid to a silk fibroin-based nerve conduit by using genipin as a crosslinking agent, and to use the TENG for repairing a 10 mm long sciatic nerve defect in rats. Our design is hoped to optimize the regenerative microenvironment by controlling the miR-17-92 cluster expression level, and to promote neurites outgrowth, facilitate Schwann cells proliferation and migration and accelerate vascular remodeling, and thus leading to the better outcome of nerve regeneration, targeted muscle reinnervation and functional recovery. This study provides some new insights into the biological processes and molecular mechanisms underlying peripheral nerve regeneration, and also contributing to the development of a potential therapeutic target for peripheral nerve injury.

探索寻找自体神经替代物用以修复周围神经缺损，一直是周围神经再生领域研究热点。本课题组利用经典的大鼠坐骨神经横断模型，不同时间点取近侧和远侧端各0.5cm神经组织进行RNA-seq深度测序，差异表达显著基因基于时序分类，结合mRNA与miRNA表达谱相关性分析，靶基因pathway，整合构建mRNA-miRNA互作网络，结果发现miR-17-92 cluster不仅是发育早期关键调控因子，同时还是周围神经再生过程关键基因。本研究拟运用组织工程研究手段，制备工程化miR-17-92 cluster过表达质粒PLGA缓释微球丝素神经移植物，并将其用于修复大鼠坐骨神经10mm缺损。通过改善局部再生微环境，促进神经元轴突生长，施万细胞增殖迁移，加速血管重建，从而加快神经生长速度，实现靶肌更快更精准重支配，达到神经功能良好恢复。本研究丰富了周围神经再生核心网络调控理论，为开展临床治疗提供新思路新方法。

周围神经损伤是临床常见病，近年来由于交通事故和各种意外伤害造成的周围神经损伤呈大幅上升趋势。周围神经损伤导致麻痹、瘫痪和丧失对相应身体区域自主控制。自体神经移植一直是修复周围神经缺损的金标准。但是由于诸多弊端，其应用受到很大限制。因此，探索寻找自体神经替代物修复周围神经缺损，一直是周围神经再生的研究热点。自体神经替代物的主要研究方向是组织工程化神经。组织工程化神经一般由支架材料、种子细胞、生长因子等几个部分构成。以生物材料导管或缓释微球凝胶为载体，负载治疗基因局部应用于损伤部位，因其安全性及相对稳定等特点，临床表现出巨大的应用前景。.MicroRNAs(miRNAs)是一类广泛存在于真核生物内源性小非编码单链RNA，参与调节各种生理病理过程。周围神经系统神经元与中枢神经系统神经元相比，其内在具有更高再生能力，在合适微环境条件下轴突能够再生。研究发现，miR-17-92cluster不仅是发育早期关键性调控因子，同时还是周围神经再生过程中的关键基因。.本研究运用组织工程学研究手段，以生物相容性和安全性更高的缓释微球为基因治疗载体，体外构建含MicroRNA-17-19cluster过表达质粒的PLGA缓释微球，体外实验证明MicroRNA-17-19cluster缓释微球能促进神经元轴突生长，促进施万细胞增殖迁移。进一步将该组织工程化神经，用于体内修复大鼠坐骨神经损伤模型。通过调控损伤局部MicroRNA-17-19cluster浓度，改善再生微环境，结果发现，MicroRNA-17-19cluster缓释微球组织工程化神经，能加快再生神经的生长速度，促进机械痛觉和温度觉的更快恢复，实现对靶肌更快更精准重支配，最终实现神经功能的良好恢复。本研究丰富了关于周围神经再生过程核心调控网络理论，有助于从新的角度解释周围神经损伤和修复机制，为将来临床治疗周围神经损伤提供新思路新方法。.