他克莫司促进神经干细胞移植大鼠脊髓损伤的再生与修复

Tacrolimus promotes repair and regeneration of injured spinal cord following neural stem cells transplantation in rats

背景:研究证实,他克莫司不仅抑制T细胞的增殖、活化,还能抑制小胶质细胞、巨噬细胞等炎症细胞在损伤局部聚集、活化及相关炎症因子的释放,减轻继发性炎症反应对原发损伤周围正常组织的破坏,从而对损伤局部的神经组织起保护作用。目的:观察他克莫司对神经干细胞移植大鼠脊髓损伤后再生修复的影响。方法:分离培养孕13dSD大鼠神经干细胞。显微镜下动脉瘤夹夹闭SD大鼠T8脊髓,建立压迫型脊髓损伤动物模型。损伤后7d随机数字表分为3组:对照组,于损伤中心定向注射生理盐水;细胞移植组,于损伤中心定向注射神经干细胞;他克莫司组,于损伤中心定向注射神经干细胞同时给予免疫抑制剂他克莫司1mg/(kg?d)腹腔注射连续7d。1,2,4,8周后,通过BDA顺行示踪、苏木精-伊红与免疫组化染色及电镜检测,观察移植后脊髓组织再生和神经元的变化。结果与结论:对照组在损伤中心端远侧无神经纤维通过。细胞移植组与他克莫司组在治疗1周后有部分神经纤维通过,8周均有部分BDA阳性标记的皮质脊髓束再生通过脊髓损伤部位,特别是他克莫司组可延续至距损伤中心1.7cm。苏木精-伊红染色显示,细胞移植组与他克莫司组2周时坏死灶开始缩小,泡沫细胞减少。电镜结果显示,他克莫司组1周时即出现较正常的微丝和微管结构,8周时星形细胞、许旺细胞、髓鞘典型多见,神经轴突的终末有较多的兴奋性递质和不典型的轴树连接,出现较多的结构正常的髓鞘。说明损伤大鼠移植神经干细胞后联合应用他克莫司后可减轻早期的急性炎症反应,保证神经细胞的存活,具有神经保护和神经营养作用,可加快神经功能的恢复。

BACKGROUND：Studies have confirmed that tacrolimus not only inhibits T cell proliferation and activation,but also suppresses microglia,macrophages and other inflammatory cells to aggregate,activate and release associated inflammatory cytokines in injuries,which can reduce the damage of secondary inflammation to surrounding normal tissue,thus,provide protective effect to nerves in injuries.OBJECTIVE：To observe the effect of tacrolimus on spinal cord regeneration and injury repair following neural stem cells （NSCs） transplantation in rats.METHODS：NSCs were isolated form 13-day-pregnant SD rats.Aneurysm clipping folder was used to clip T8 spinal cord of rats under microscope to establish animal model of spinal cord compression injury.At 7 days after injury,all rats were randomly divided into 3 groups：rats in the control group were injected with normal saline at the injury center orientation;those in the cell transplantation group received center directional injection of neural stem cells （NSCs）;those in the tacrolimus （FK506） group received directional injection of NSCs combined with intraperitoneal injection of immunosuppressant FK506,1 mm/kg per day for 7 continuous days.The spinal cord tissue regeneration and neuronal changes were observed using BDA anterograde tracer,haematoxylin-eosin staining,immunohistochemistry and electron microscopy at 1,2,4 and 8 weeks after operation.RESULTS AND CONCLUSION：There were no fibers passing through the distal end of the injury center in the control group.However,some nerve fibers in the cell transplantation and FK506 groups passed at 1 week after treatment.Parts of regenerating BDA-positive corticospinal tract passed through the site of spinal cord injury in the cell transplantation and FK506 groups at 8 weeks after treatment,especially in the FK506 group,which may be continued to 1.7 cm of the injury center.Hematoxylin-eosin staining showed that,the necrotic foci began to shrink,and the foam cells decreased in the cell transplantation group and FK506 group at 2 weeks after treatment.Electron microscopy results showed that,in the FK506 group,more normal microfilament and microtubule structure appeared at 1 week,the astrocytes,Schwann cells,and myelin sheaths were commonly seen at 8 weeks after treatment,nerve axons of excitatory terminals had more excitatory transmitter and non-typical tree-axis connections,and there were more normal myelin sheaths.It illustrated that,NSCs transplantation combined with FK506 can reduce the early post-acute inflammatory response,ensure the survival of nerve cells,exhibit neuroprotective and neurotrophic roles,as well as speed up the recovery of neurological function.