同种异体神经复合体桥接修复兔坐骨神经缺损与神经-肌结构重建及功能恢复

Recellularized neural complex for repair of sciatic nerve gap and nerve-muscle construction as well as functional rehabilitation in rabbits

背景:课题组在前期研究中分别探讨了神经生长因子对胎兔许旺细胞培养的影响以及去细胞神经移植体的制备,并在体外成功制备出重新细胞化的神经移植复合体。目的:探讨种植许旺细胞的去细胞同种异体神经移植对神经-肌结构重建和功能恢复的作用。设计、时间及地点:随机对照动物实验,于2006-03/2007-06在河北医科大学第三医院中心实验室完成。材料:健康成年新西兰白兔37只,1只用于制备去细胞神经桥接体,剩余36只随机分为实验组、对照组,18只/组。方法:切取兔双侧坐骨神经,剥除周围组织,剪成约每段3cm,放入TritonX-100水溶液中静置12h,蒸馏水漂洗,以使溶于水的TritonX-100膜蛋白体复合物充分脱离神经干,如此反复,TritonX-100作用时间共为96h,萃取好的去细胞神经桥接体在Hank's液中4℃保存。调整第2代许旺细胞浓度至1×1011L-1,用100μL微量注射器注入去细胞神经桥接体内,然后将神经段置于DMEM培养液中,即为同种异体神经复合体。实验组兔于膝关节上方造成20mm长缺损,将种植许旺细胞的同种异体神经复合体缝接于坐骨神经两断端;对照组兔左侧坐骨神经造成20mm长缺损后,用单纯的去细胞同种异体神经桥接物缝接神经两断端。主要观察指标:分别于术后4,8,16周大体观察兔足部溃疡形成及愈合情况,通过肌电图、光镜、电镜等方法检测远端腓神经的轴突、髓鞘及胫骨前肌、运动终板的恢复情况。结果:术后4,8,16周两组动物术区局部均未出现明显的排斥反应,实验组足部溃疡愈合情况、神经纤维数目、髓鞘及再生神经的超微结构、运动终板的形态及靶肌肉结构的恢复均优于对照组。术后4周两组均未见明显的神经传导,术后8,16周实验组胫骨前肌湿质量、神经传导速度均优于对照组(P<0.05)。结论:种植许旺细胞的去细胞同种异体神经复合体不仅能够为再生神经提供良好的支架作用,还能诱导和促进神经轴突和髓鞘的再生,对坐骨神经缺损后的神经-肌结构重建与功能恢复具有显著的促进作用。

BACKGROUND： Previous studies has explored the effects of nerve growth factor （NGF） on cultured rabbit Schwann cells as well as preparation of acellular nerve allografts, in addition, the neural complex was prepared in vitro. OBJECTIVE： To investigate the effect of acellular nerve allografts on the functional recovery and reconstruction of the nerve-muscle structure of the sciatic nerve defect in rabbits. DESIGN, TIME AND SETTING： The randomized controlled animal experiment was performed at the Center Laboratory of Third Hospital of Hebei Medical University from March 2006 to June 2007. MATERIALS： Totally 37 healthy, adult, New Zealand, white rabbits were selected. One rabbit was prepared for extracted nerve bridge, and the others were randomly divided into the experimental and control groups, with 18 animals in each group. METHODS： The double sides of sciatic nerve were removed, divested surrounding tissues, and cut into 3 cm segments, then placed in TritonX-100 solution for 12 hours, washing with distilled water. The procedure was repeated, and TritonX-100 totally used for 96 hours. Then the extracted nerve bridge was conserved in Hank＇s solution at temperature of 4℃. The concentration of 2^rd passage Schwann cell was regulated to 1×10^11/L, and injected to extracted nerve bridge, followed by DMEM culture, to obtain neural complex. Rabbits in the experimental group were prepared a 20 m defects above knee joints, and the recellularized neural complex were transplanted into the defective sciatic nerve. Rabbits in the control group were prepared by transplanting extracted nerve bridge without Schwann cells. MAIN OUTCOME MEASURES： The elcosis and healing of feet were observed at weeks 4, 8, and 16 after operation, meantime, the recovery of axon of distal end peroneal nerves, myelin sheath, tibialis anterior, as well as motor end plate were detected by electromyogram, light microscope and transmission electron microscope. RESULTS： At weeks 4, 8 and 16 after operation, the rejection was not found in the operating field. The experimental group was better than the control group in healing of ulcers, the number of nerve fibres, the ultramicrostructure of medullary sheaths, the regeneration nerve, the structure of the motor end plate and muscle. At 4 weeks after operation, no nerve conduction was found in 2 groups, but at weeks 8 and 16 after operation, the experimental group was better than the control group in wet weight of tibialis anterior muscle and nerve conduction velocity （P 〈 0.05）. CONCLUSION： The recellularized neural complex can significantly promote the reconstruction of the nerve-muscle structure and functional recovery of injured sciatic nerve.