许旺细胞修复大鼠脊髓损伤:静脉移植的可行性及优势

背景:目前细胞移植是修复脊髓损伤的研究热点之一,许旺细胞能够分泌多种神经营养因子,改善损伤局部微环境,大量相关文献证实了其能够促进脊髓损伤后功能的恢复.针对治疗脊髓损伤的细胞移植方法很多,其中静脉移植具有操作方便、能够避免传统移植方法造成的附加损伤等优点.目的:探讨许旺细胞尾静脉移植修复大鼠脊髓损伤的疗效.方法:体外分离Wistar大鼠双侧坐骨神经,植块法培养,S-100染色鉴定,Hoechst33342标记许旺细胞.Impactor model-Ⅱ打击仪制作 Wistar大鼠T10脊髓损伤模犁60只,随机分为3组,空白对照组术后未作处理,DMEM对照组、许旺细胞移植组术后1周分别尾静脉注射1 mL.DMEM或1 mL.许旺细胞.术前1 d,术后1,3 d,1周及以后每周进行BBB评分.移植后1,2,4周取材行冰冻切片观察移植许旺细胞的迁移.移植后8周取材行苏木精-伊红染色及免疫荧光染色观察损伤段胶质原纤维酸性蛋白、神经元特异性烯醇化酶的表达.结果与结论:经纯化鉴定获得纯度为95%的许旺细胞.移植后1,2,4周损伤段及邻近节段脊髓内可观察到Hoechst33342阳性细胞.术后4周,许旺细胞移植组与空白对照组、DMEM对照组BBB评分差异开始有显著性意义(P<0.05),且许旺细胞移植组高于空白对照组、DMEM对照组.苏木精-伊红染色示各组均有脊髓空洞形成,许旺细胞移植组空洞小于空白对照组、DMEM对照组.免疫组织化学染色示空白对照组、DMEM对照组胶质原纤维酸性蛋白反应较强,许旺细胞移植组胶质原纤维酸性蛋白阳性面积小于其他组(P<0.05);许旺细胞移植组神经元特异性烯醇化酶的阳性面积明显大于其他组(P<0.05).提示静脉移植许旺细胞修复大鼠脊髓损伤是可行的,操作简便,且有较好的疗效.

Repair of spinal cord injury using Schwann cells in rats: Feasibility and superiority of intravenous transplantation

BACKGROUND: Emerging studies have focused on cell transplantation. Schwann cells (SCs) can secrete various neurotrophic factors and improve local environment around injury. Plenty of documents have demonstrated that SCs could promote functional recovery following spinal injury. Many transplanting methods are available for treating spinal cord injury, and the intravenous cell transplantation is profitable for easy operation and avoidance of additional trauma. OBJECTIVE: To investigate the effects of intravenous transplantation of SCs on spinal cord injury in rats. METHODS: The bilateral sciatic nerves of Wistar rats were separated in vitro, cultured by tissue clot method, identified by S-100 and labeled by Hoechst33342. Sixty rat models with T10 spinal cord injury were prepared using impactor model- II type weight drop apparatus. Then the injured rats were randomly divided into 3 groups: blank control, DMEM control and SCs transplantation groups. No treatment was performed in the blank control group. Totally 1 mL DMEM and or SCs was injected into rats of DMEM control and SCs transplantation groups by tail vein respectively. Basso Beattie Bresnahan (B6B) scores were performed at 1 day before and 1, 3 days, 1 week and weekly after operation. The migration of transplanted SCs was observed at 2 weeks and 4 after transplantation. The expressions of glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) were detected by haematoxylin-eosin staining and immune-fluorescence staining.RESULTS AND CONCLUSION: The purity of SCs reached 95%. Hoechst33342 positive cells were observed throughout the injured and the nearby region of spinal cord at 1, 2, and 4 weeks after transplantation. The statistical difference of BBB score among the SCs transplantation, blank control, and the DMEM control groups displayed at 4 weeks after transplantation (P < 0.05), and the BBB scores of the SCs transplantation were higher than other groups. Haematoxylin-eosin staining showed the cavity formed in each group at 8 weeks after transplantation, but the area of SCs transplantation was smaller than that of the blank control and DMEM control groups. The immunofluorescence staining indicated that the expression of GFAP were more intense in the blank control group and DMEM control than SCs transplantation (P < 0.05), while the expression of NSE was more intense in SCs transplantation than other groups (P< 0.05). It implied that intravenous transplantation of SCs promotes regeneration of axon and improves neurological functions after spinal cord injury in rats.