自体成肌细胞修复周围神经缺损

Autologous myoblasts for repairing peripheral nerve defect

目的:观察自体成肌细胞在神经再生室中的作用,探讨其作为组织工程的种子细胞修复周围神经缺损的可行性。方法:实验于2002-08/2003-08在北京军事医学科学院基础医学研究所九室完成。①成年Wistar大鼠30只,采用随机数字法分成成肌细胞组、细胞外基质凝胶组和生理盐水组,每组10只。②切除大鼠右坐骨神经6mm,断端分别套入硅胶管,使神经两断端在硅胶管内相距13mm。③成肌细胞组:快速分离骨骼肌成肌细胞,差速贴壁法纯化、增殖,按1×109L-1细胞浓度与细胞外基质凝胶混合,植入神经再生室内;细胞外基质凝胶组:神经再生室内植入细胞外基质凝胶;生理盐水组:神经再生室内植入生理盐水。④术后4,8,12周进行大鼠坐骨神经功能指数测定;术后12周进行大体观察、电生理检测、腓肠肌湿重、神经组织学及超微结构观察。结果:30只大鼠全部进入结果分析。①结蛋白免疫组织化学:阳性细胞平均达98.01%。②大体观察:术后12周,成肌细胞组和细胞外基质凝胶组均可见再生神经,外形似正常神经,直径较正常的神经干细,成肌细胞组略粗于细胞外基质凝胶组。生理盐水组导管内无再生神经通过间隙。③坐骨神经功能指数:术后8,12周,成肌细胞组显著高于细胞外基质凝胶组(8周:-71.788±3.569,-76.986±2.266;12周:-61.847±2.914,-69.527±1.765;P均<0.01)。④电生理检查:术后12周,成肌细胞组的运动神经传导速度及波幅明显高于细胞外基质凝胶组,成肌细胞组、细胞外基质凝胶组腓肠肌肌电图呈部分失神经电位表现,生理盐水组则为完全失神经电位。⑤腓肠肌湿重恢复率:成肌细胞组明显好于细胞外基质凝胶组和生理盐水组。⑥组织学检查:术后12周,成肌细胞组、细胞外基质凝胶组的再生神经纤维排列整齐、密集,神经导管交界处无瘢痕,成肌细胞组的再生神经纤维多且直径较粗大,排列更为规则。⑦透射电镜观察:成肌细胞组和细胞外基质凝胶组均见再生的有髓神经纤维。⑧再生神经纤维图像分析:术后12周,在有髓神经纤维密度、神经纤维有效面积、有髓神经纤维数量、直径及髓鞘厚度等指标上成肌细胞组均优于细胞外基质凝胶组。结论:自体成肌细胞能够促进周围神经再生,可以作为种子细胞应用于外周神经组织工程。

AIM： To observe the effect of autologous myoblasts in nerve regeneration chambers and explore the feasibility of autologous myoblasts as seed cells for nerve tissue engineering to repair peripheral nerve defects. METHODS： The experiment was performed at the Ninth Room of Institute of Basic Medical Sciences, Beijing Academy of Military Medical Sciences from August 2002 to August 2003. ①Totally 30 adult Wistar rats were divided into myoblast group, extracellular matrix gel group and saline group with 10 ones in each group. ②A 6 mm-long nerve defect was made in the right sciatic nerve and was bridged by a silicone tube at broken ends of fractured bone, respectively. The distance between the two broken ends was 13 mm in silicone tube. ③Myoblast group： Skeletal muscle sarcoblast was isolated rapidly. Sarcoblast was purified and proliferated with different velocity adherence method at 1×10^9 L^-1, and then grafted into neural regeneration chamber. Extracellular matrix gel group： Extracellular matrix gel was implanted into neural regeneration chamber. Saline group： Neural regeneration chamber was filled with saline. ④ Sciatic nerve function index was detected at week 4, 8 and 12 after operation. Gross observation, electrophysiological exam, wet weight of gastrocnemius muscle, neurohistology and uhrastructure were observed at week 12 after operation. RESULTS： Totally 30 rats were involved in the result analysis. ①Desmin immunohistochemistry stain： Positive cells reached 98.01% averagely.② Gross observation： After 12 week of operation, there was much regeneration nerve fiber in myoblast group and extraceltular matrix gel group, like normal nerve; The diameter was thinner than normal nerve trunk, it was slightly thicker in the myoblast group than that in the extracellular matrix gel group. There was no regeneration nerve through gap in saline group. ③ Sciatic nerve function index： After 8, 12 week of operation, myoblast group had higher score than extracellular matrix gel group （week 8：-71.788±3.569, -76.986 ±2.266; week 12：-61.847 ±2.914, -69.527 ± 1.765 ; P 〈 0.01 ）. ④ Electrophysiologic study： After 12 week of operation, the motor nerve conduction velocity and wave amplitude of myoblast group were significantly higher than those in the extracellular matrix gel group. The gastrocnemius electromyogram showed partly absent neuropotential in myoblast group and extracellular matrix gel group, but saline group being completely absent neuropotential. ⑤Recovery rate of gastrocnemius wet weight： The myoblast group was better than extraeellular matrix gel group and saline group. ⑥ Histochemistry examination： After 12 week of operation, the regeneration nerve fiber of mynblast group and extracellular matrix gel group lined up in order and intensive. There was not scar in nerve-tube juncture, and the regeneration nerve fiber of myoblast group was thicker and more orderly. ⑦Observation with transmission electron microscope： There were regeneration medultated nerve fibers in myoblast group and extracellular matrix gel group. ⑧Analysis of regeneration nerve fiber image： After 12 week of operation, effective area of nerve fiber, the medullated nerve fibers density, quantity, diameter and neural sheath thickness of myoblast group were all better than those of the extracellular matrix gel group. CONCLUSION： Autologous myoblasts can promote peripheral nerve regeneration and can be used as seed cells for peripheral nerve tissue engineering.