不同途径移植自体激活雪旺细胞修复大鼠脊髓损伤

目的 通过不同途径移植自体激活雪旺细胞(autologous activated Schwann cells,AASCs),评价各种移植途径在修复脊髓损伤中的作用.方法 结扎Wistar大鼠双侧隐神经,1周后取下结扎处远端神经,在体外分离、培养、传代、纯化和鉴定后获得供实验用AASCs.60只Wistar大鼠制成T_(10)脊髓损伤模型后随机分为三组,1周后将预先用Hoechst33342标记的AASCs移植到三组大鼠体内:Ⅰ组,尾静脉移植;Ⅱ组,鞘内移植(经蛛网膜下腔);Ⅲ组,局部损伤处移植.术后采用BBB评分评价大鼠功能恢复.3个月后行BDA皮质脊髓束顺行示踪标记.标记2周后处死动物,取出损伤处脊髓行快速冰冻切片,行Cy3荧光探针染色、神经丝蛋白200(NF200)和HE染色.结果 AASCs在体外可稳定传4代以上,并表达S-100抗原.从术后第4周开始,BBB评分在各组间差异有统计学意义.至实验结束时,HE染色显示Ⅲ组中损伤空洞明显小于其余两组,NF200免疫组化染色阳性面积占总面积百分比各组间差异有统计学意义.BDA神经示踪显示,Ⅲ组中有较多的再生轴突通过脊髓损伤区,横断面上再生轴突的免疫组化阳性面积各组间差异有统计学意义.结论 局部损伤处移植AASCs可以有效保证移植细胞的数量,AASCs通过分泌多种营养因子和桥接损伤轴突再生的作用促进大鼠脊髓损伤后的功能恢复.

Treatment of rat spinal cord injury with autologous activated Schwann cells via different grafting routes

Objective To explore the curative effect of autologous activated Schwann cells (AASCs) in rat spinal cord injury (SCI) via different grafting routes. Methods Saphenous nerve of the adult Wistar rat was ligated for one week to activate Schwann cells. And then AASCs were cultured, passaged, purified and identified in vitro. Sixty Wistar rats were all made into SCI model at T_(10) and then divided into three groups randomly. One week after injury, AASCs were transplanted into the SCI rats via three different routes group Ⅰ: tail vein; group Ⅱ: intrathecal (subarachnoid route); group Ⅲ: intraspinal cord]. BBB score was carried out to evaluate the functional recovery of the injured rats at weekly interval postinjury. Three months later, corticospinal tract (CST) was labeled by 10% BDA. Two weeks after labeling, the rats were sacrificed and then their injured spinal cord tissues were take out to carry out 10 μm fast frozen section, which was then followed by Cy3 fluorescent probe staining, NF200 and HE staining. Results AASCs passed over 4 stable passages and expressed S-100 antigen in vitro. The difference among groups could be considered sig-nificant in BBB score on the 5th week postinjury. HE staining showed that the injured cavity was evidently smaller in group Ⅲ compared with other two groups. Statistical difference also existed among groups by com-paring the percentage of positive response areas in NF200 staining. 10%BDA stain demonstrated that more regenerated axons passed through the injured cavity in group Ⅲ when compared with other two groups, which was also confirmed by immunohistochemistry stain. Conclusion The amount of grafted AASCs can be guaranteed effectively in injured epicenter via the grafting route of intraspinal cord. AASCs promote the repair after SCI by secreting multiple kinds of neurotrophic factors and bridge injured axons for regrowth.