神经营养因子-3基因移植的雪旺细胞延缓肌萎缩的实验研究

目的 探讨神经营养因子-3(neurotrophic factor-3,NT-3)基因修饰的雪旺细胞(Schwann cells,SC)延缓失神经性肌肉萎缩的作用.方法 采用双酶消化法和贴壁法培养、纯化与传代SC.应用阳离子脂质体以NT-3基因修饰SC,免疫组织化学S-100染色检测NT-3基因转入前后SC的纯度.切断右侧胫神经建立腓肠肌失神经支配的动物模型.将104只SD大鼠按注射药物的不同随机分为4组,每组26只.A组,细胞外基质(extracellular matrix,ECM)凝胶组;B组,SC-ECM凝胶组;C组,NT-3基因-ECM凝胶组;D组,NT-3基因修饰的SC-ECM凝胶组.术后12周进行腓肠肌肌肉电生理,8周和12周做肌湿重、肌纤维横截面积的检测.结果 NT-3转染前后SC纯度分别为(94.7±2.1)%及(95.6±2.5)%,两者比较差异有统计学意义(P＜0.05).术后12周用电刺激腓肠肌,均可引出肌肉收缩活动;且随着时间的延长,单次收缩的波幅、速度,及强直收缩的时间和强直收缩波幅的恢复率均增加.D组均优于B、C组,B、C组均优于A组(P＜0.05),而B、C组相比差异无统计学意义(P＞0.05).术后8周和12周的肌湿重与肌纤维横截面积D组均优于B、C组,B、C组均优于A组(P＜0.05),而B、C组相比差异无统计学意义(P＞0.05).结论 转染NT-3基因的SC移植能够实现失神经骨骼肌的神经再支配,并且能与骨骼肌建立起功能性突触连接,有延缓失神经性骨骼肌萎缩的作用.

Prevention of denervation muscle atrophy by transplantation of neurotrophic factor-3 (NT-3) modified Schwann cells (SC) in rats

Objective To explore the effects of neurotrophic factor-3 (NT-3) modified Schwann cells transplantation on preventing atrophy of denervated skeletal muscles in rats. Methods Schwann cells were isolated, cultured and purified using double enzyme digestion and time-speed difference method. The NT-3 cDNA gene was transfected into the cultured Schwann cells by using cationic liposome. Schwann cells were identified through the phase contrast microscope and S-100 immunohistochemical staining after being modified.Gastrocnemius muscle denervation model was set up by transecting the right tibial nerve in 104 adult SD rats,which were randomly divided into4 groups, with 26 each. Extracellular matrix (ECM) matrigel loaded with or without NT-3 and SC was injected into the distal tibial nerve according to group assignment. In group A plain matrigel was injected. In group B matrigel with Schwann cells was injected. In group C matrigel with NT-3 gene was injected. In group D matrigel with NT-3 modified Schwann cells was injected. At 8 and 12 weeks postoperatively, reinnervation of denervated skeletal muscle was determined by electrophysiology, muscle wet weight and muscle fiber cross-sectional area examination. Results The purity of SC was(94.7±2.1)% and (95.6 ± 2.5)% before and after NT-3 modification, respectively. The difference was statistically significant (P ＜0.05). At 8 and 12 weeks postoperatively, contraction of the gastrocnemius muscle could be elicited by electric stimulation in groups B, C and D but not in group A. Force and velocity of twitch tension and force and duration of tetanic tension of the gastrocnermius muscle increased as postoperative interval prolonged. These parameters were better in group D than in groups B and C, and better in groups B and C than in group A (P ＜0. 05). The difference between group B and C was not significant ( P ＞ 0.05). Muscle wet weight and muscle fiber cross-sectional area were higher in group D than in groups B and C, and higher in groups B and C than in group A ( P ＜ 0.05). The difference between group B and C was not significant ( P ＞ 0.05). Conclusion Transplantation of NT-3 gene modified Schwann cells into the peripheral nerve can promote nerve and axonal regeneration, reinnervate skeletal muscle and form neuromuscular junctions to effectively prevent muscle atrophy.