骨髓源性神经干细胞周围神经移植的实验(英文)

Experiment on graft of bone marrow-derived neural stem cells in peripheral nerve

背景:由于周围神经只是神经元细胞的轴突,有关神经干细胞的研究几乎都集中在神经元细胞上,因此,关于周围神经修复的研究可以借鉴神经干细胞研究的某些经验。目的:观察自体骨髓源性神经干细胞于周围神经损伤区移植后周围神经修复情况,以及所移植神经干细胞能否在周围神经损伤区以分化神经元的形式存活并迁移到脊髓。设计:观察对比实验。单位:南方医科大学珠江医院神经医学研究所。材料:清洁级新西兰大白兔8只,体质量1.5～2.5kg,雌雄不拘。方法:实验于2004-04/10在南方医科大学珠江医院神经医学研究所完成。①取新西兰大白兔骨髓基质细胞体外培养及诱导分化为神经干细胞。②采用以溴脱氧尿嘧啶核苷标记细胞和免疫细胞化学SABC法检测观察培养细胞。③制备兔坐骨神经损伤模型,随机选择一侧坐骨神经损伤区为移植侧,注入生理盐水、胶原基质蛋白及细胞包埋液至0.01mL(含干细胞1×1010L-1);另外一侧为对照侧,注入胶原基质蛋白悬液0.01mL。移植后3个月灌注固定,行自体周围神经损伤区移植;移植区、脊髓区、对侧损伤区和正常神经区取材观察。主要观察指标:神经干细胞移植区、脊髓区、对侧损伤未移植区神经纤维形态及神经元细胞。结果:移植区生长的神经纤维密度及连续性明显比对侧未移植区高,光镜下可见较多的许旺细胞;放大至400倍视野下,还可见有髓神经纤维的郎飞氏结;神经纤维间隙也可见有黏液基质,并有少数成纤维细胞;移植区、脊髓区和对侧未移植坐骨神经损伤区均未见有神经元样细胞存在。结论:自体骨髓源性神经干细胞周围神经缺损区移植有利于神经纤维的修复,不能在周围神经移植区、脊髓区和对侧缺损对照区以神经元的形式存活。

BACKGROUND： Since there are only cell axons of neurons in peripheral cells, the study on neural stem cells （NSCs） is almost focused on neuronal cells, for which, the study on repair of peripheral nerve may be based on some experiences in NSCs. OBJECTIVE： To observe the repair of peripheral nerve after graft of autologus bone marrow derived NSCs in the injured area. To observe whether the grafted NSCs were survived and migrated in spinal cord as differentiated neurons in the injured area of peripheral nerve or not. DESIGN： Observed controlled experiment was designed. SETTING： Institute of Neurological Medicine of Zhujiang Hospital affiliated to Southern Medical University MATERIALS： Eight New Zealand big white rabbits were employed, of clean grade, mass weighted varied from 1.5 to 2.5 kg and of either sex. METHODS： The experiment was performed in Institute of Neurological Medicine of Zhujiang Hospital affiliated to Southern Medical University from April to October 2004. ① Bone marrow stromal cells （BMSCs） were collected from New Zealand big white rabbits for culture and differentiation of NSCs in vitro. ② BrdU labeling and immunocytochemieal SABC assay were used to observe cultured cells. ③ Rabbit model with sciatic injury was prepared. Sciatic neural injured area of one side was randomized as graft side. Physiological saline, collagen matrix and cellular embedding solution were infused up to 0.01 mL （containing stem cells 1×10^10L^-1）. Another side was taken as the control, in which, collagen matrix suspension 0.01 mL was infused. Perfusion and fixation were followed 3 months after graft and auto-graft was performed in the injured peripheral nerve. The materials were collected for observation from graft area, spinal cord area, injured area on the opposite side and normal neural area. MAIN OUTCOME MEASURES： Morphology of nerve fibers and neuronal cells in NSC graft area, spinal cord area and non-graft area on opposite injury side. RESULTS： The density and continuity of nerve fibers grown in graft area were higher remarkably than non-graft area on opposite side and more Schwann ceils were seen under optic microscope. With amplified ×400 visual field, Ranvier＇s node of spinal nerve fiber was visible. In addition, mucous matrix and few fibroblasts were seen also in the space of nerve fibers. The survived neuronal cells were no visible in graft area, spinal cord area and non-graft area of sciatic injury on the opposite side. CONCLUSION： Graft of autologus bone-marrow derived neural stem cells in defect area of peripheral area benefits repair of nerve fibers. But the neural stem cells cannot survive as neurons in graft area of peripheral nerve, spinal cord area and the defect control on the opposite side.