去细胞神经支架联合表皮神经嵴干细胞源性雪旺细胞修复面神经长距离缺损的实验研究

Acellular nerve grafts made through chemical extraction have incomparable and irreplaceable advantages over other materials in bridging large facial nerve defects, such as trunk and multiple branches region of extratemporal segment, and acellular nerve allografts (ANAs) have chieved satisfactory results clinically. Recently, acellular nerve xenografts (ANXs) have become the new research topic. Our previous experiments indicated that ANXs could repair facial nerve defects,ANAs combined with Schwann cells could promote better effects on repairing large facial nerve defects than ANAs alone in animal model, and epidermal neural crest stem cells(EPI-NCSCs) from hair follicles can be differentiated into Schwann cells. Thus we infer whether EPI-NCSCs can be implanted into ANXs, followed by repairing large facial nerve defects, which will help to solve the problem of lack of appropriate repair material. In order to provide experimental basis for clinical application of ANXs combined with EPI-NCSCs in repairing large facial nerve defects, in the present study we will take the rhesus monkey as the experimental object, and detect nerve electrophysiology, retrograde trace of facial nerve motor neuron, ultrastructure of the regenerated nerve and histology of the target muscle, as well as the immunological rejection and the growth and outcome of EPI-NCSCs labeled with green fluorescent protein in vivo postoperatively.

化学萃取法制备的去细胞神经在修复面神经特殊部位（如颞骨外段主干-多分支处）长距离缺损具有其他移植材料难以比拟和替代的优势。去细胞异体神经已经取得了良好的临床效果，近来去细胞异种神经成为新的研究热点。申请人前期研究发现去细胞异种神经可桥接面神经缺损，植入雪旺细胞可增强去细胞神经支架的修复效果，毛囊表皮神经嵴干细胞可分化为雪旺细胞。由此推断采用表皮神经嵴干细胞源性雪旺细胞植入去细胞异种神经修复面神经长距离缺损，可能有助于解决临床缺乏合适修复材料的难题。为了验证这一假说，本课题拟用去细胞异种神经支架联合表皮神经嵴干细胞修复猕猴面神经主干-多分支处缺损，术后通过检测神经电生理、运动神经元逆行示踪、再生神经超微结构、靶肌肉组织等观察神经再生效果，并与去细胞异种神经及自体神经移植对照；同时观察免疫排斥反应，以及表皮神经嵴干细胞源性雪旺细胞在体内的生长和转归情况，为面神经缺损修复提供新的可能替代材料。

研究背景：化学萃取法制备的去细胞神经在修复全面神经缺损（涉及颞骨外段主干-多分支处）长距离缺损具有其他移植材料难以比拟和替代的优势。课题负责人前期研究发现去细胞异种神经可桥接面神经缺损，植入雪旺细胞可增强去细胞神经支架的修复效果，大鼠毛囊表皮神经嵴干细胞可分化为雪旺细胞。.研究内容：本课题应用改良Sondell法制备细长电缆状去细胞异种神经支架（组织学、体内安全性检测），联合表皮神经嵴干细胞源性雪旺细胞（P75和P63鉴定干细胞，S100和 GFAP鉴定雪旺细胞）修复猕猴全面神经缺损，术后通过观察面部表情（静、动态，β角测量）、检测神经电生理（潜伏期、波幅恢复情况、眼-颊联带神经电生理）、运动神经元双重逆行示踪、再生神经组织学观察及超微结构、靶肌肉组织等观察神经再生效果，同时观察免疫排斥反应（外周血淋巴细胞，外周血细胞因子，再生神经淋巴细胞及巨噬细胞），以及移植细胞在体内的生长和转归情况（EdU标记移植细胞）。.结果：（1）改良Sondell法制备支架的形态保存以及体内安全性好。（2）成功分离、培养、鉴定猕猴毛囊表皮神经嵴干细胞，无基因修饰下可转化为雪旺细胞。（3）支架对细胞无毒性，相容性好。（4）面神经缺损修复后联带运动可通过眼-颊联带神经电生理、双重荧光剂逆行示踪进行检测。（5）面神经长距离缺损修复，与单纯支架移植相比，联合细胞移植可增强支架的部分神经再生修复效果，包括复合肌肉动作电位（CAMPs），再生神经纤维数量、直径、髓鞘厚度等，但未能减少联带运动的发生和强度。（6）面神经长距离缺损修复，与自体神经移植相比，细长电缆状去细胞神经支架或复合毛囊表皮神经嵴干细胞源性雪旺细胞未引起明显的局部/全身免疫反应。（7）EdU标记可用于细胞在活体内长时间存活、增殖及迁移的观察。.科学意义：去细胞神经支架联合表皮神经嵴干细胞源性雪旺细胞为面神经长距离缺损修复提供新的可能替代材料。