Dhh基因增强组织工程化神经移植修复周围神经缺损的研究

Peripheral nerve injury is a common case in clinic. Treatment on the injury of long distance is still unsatisfactory and causes difficulties for people at present. Recent studies showed that perineurial cells extended to the injury site quickly after peripheral nerve injury. Perineurial cells participated in the formation of the cellular bridge earlier than Schwann cells, and they played an important role in nerve regeneration. Desert hedgehog (Dhh) is a secreted protein which plays a key role in the formation of perineurium. However, whether the effect of Dhh on perineuium could promote nerve regeneration still needs to be elucidated.. In the present study, lentiviral vectors expessing Dhh gene are to be used to make Dhh gene overexpression in hfNCSCs. The co-culture system of Dhh-hfNCSCs, neurons and perineurial cells will be established. Then, an heterogeneous acelluar basal lamina tube seeded with Dhh – hfNCSCs, that is, the Dhh gene strengthened tissue engineered nerve is to be used for transplantation in vivo to repair a long sciatic nerve defect. Methods of morphology, molecular biology, functional evaluation and so on will be used to mainly explore the effect of Dhh – hfNCSCs on the growth, proliferation, and migration of perineurial cells in vitro and in vivo. And to cast light upon the influence of such effect on nerve regeneration and nerve functional recovery. The present study will achieve multiple cell mobilization and provide a new way for repairing long peripheral nerve defect.

周围神经损伤是临床常见病例，目前对于长距离周围神经缺损的治疗仍是个难题。近年的研究显示周围神经损伤后，神经束膜细胞快速向损伤部位延伸，早于施万细胞参与细胞桥的形成，对神经再生有重要作用。沙漠刺猬因子（Dhh）是一种分泌蛋白，在调节神经束膜的形成方面起着关键作用，而Dhh对神经束膜的调节作用是否能促进神经再生尚不明确。本项目拟以慢病毒为载体，在hfNCSCs中过表达Dhh，建立Dhh-hfNCSCs与神经元、神经束膜细胞的共培养模型；将Dhh-hfNCSCs与异种去细胞神经基膜管相复合，在体外构建Dhh基因增强组织工程化神经，移植修复长距离坐骨神经缺损。拟采用形态学、分子生物学和神经功能检测等研究方法，重点研究Dhh–hfNCSCs在体外、体内对神经束膜细胞的生长、增殖、迁移的作用，并阐明此作用对神经再生和神经功能恢复的影响。以期实现多种细胞的动员，为长距离周围神经缺损的修复提供新途径。

周围神经损伤是临床常见病例，目前对于长距离周围神经缺损的治疗仍是个难题。近年的研究显示周围神经损伤后，神经束膜细胞快速向损伤部位延伸，早于施万细胞参与细胞桥的形成，对神经再生有重要作用；毛囊神经嵴干细胞（hfNCSCs）具有神经干细胞的特性，对于神经损伤修复具有促进作用；沙漠刺猬因子（Dhh）是一种分泌蛋白，在调节神经束膜的形成方面起着关键作用。本项目成功培养了hfNCSCs和神经束膜细胞，以慢病毒为载体，在hfNCSCs中成功过表达Dhh，通过Transwell小室建立了hfNCSCs与神经束膜细胞的共培养模型，观察到hfNCSCs在共培养18h时较为明显地促进了神经束膜细胞的迁移；CCK8法检测到Dhh蛋白在神经束膜细胞的生长早期对其增殖有明显的促进作用；将Dhh-hfNCSCs与猪去细胞神经基膜管相复合，在体外构建了Dhh基因增强组织工程化神经，移植修复大鼠坐骨神经缺损。本项目为实现多种细胞的动员、修复长距离周围神经缺损提供了新途径。