Etanercept联合转TrkC基因神经干细胞移植治疗大鼠脊髓损伤

Nowadays, there are a lot of treatments for spinal cord injury, but it still has much room for improvement. In our previous study, Etanercept was administered by intraperitoneal injection 1 hour after thoracic spinal cord injury in rats.Our results indicated that etanercept reduces the associated tissue damage of spinal cord injury, improves hindlimb locomotor function, and facilitates myelin regeneration. However, rats still had significant motor disturbances in the hind limb 8weeks after Etanercept treatmant campared with normal control group. Then it is unclear whether the inhibition of inflammatory response following spinal injury combined with neural stem cells (NSCs) transplantation can improve the recovery of spinal cord injury. Therefore, we speculate that the combination therapy of Etanercept and transplantation of TrkC gene-transfected NSCs will obtain better outcomes than a single treatment. In the present study, we intend to perform spinal cord contusion models in rats, and intraperitoneally inject Etanercept 1h after the contusion injury, and administrate the transplantation of TrkC gene-transfected NSCs with local injection of NT-3 7 days after spinal cord injury. The BBB scale is used to evaluate the rat locomotor function after combination therapy. The expression of inflammatory cytokines and apoptosis-related proteins is examined by RT-PCR, western blot, and immunohistochemical analyses after treatment. Myelin regeneration is also compared by hematoxylin-eosin and luxol fast blue staining. This study will help elucidate the effect mechnisms of the combination therapy of Etanercept and transplantation of TrkC gene-transfected NSCs, and provide new ideas and methods for further research in the treatment of secondary spinal cord injury.

脊髓损伤目前治疗手段众多，但治疗效果仍有待改进。我们前期研究结果发现：大鼠急性脊髓损伤后1 h应用Etanercept可抑制神经元和少突胶质细胞凋亡、促进神经元和髓鞘再生，但治疗8周后大鼠下肢功能较正常对照组仍有明显差距。那么，在控制损伤后炎症反应的同时联合NSCs移植，是否能够提高脊髓损伤的治疗效果，目前尚不明确。我们推测，Etanercept联合TrkC基因修饰NSCs移植治疗脊髓损伤，可以取得较单一治疗方法更好的治疗效果。本研究拟制备大鼠脊髓挫伤模型，早期应用Etanercept腹腔注射，损伤后7天给予TrkC基因修饰NSCs移植和NT-3局部注射，观察大鼠脊髓损伤治疗前后运动功能恢复情况，比较联合治疗与单一治疗的疗效，检测治疗前后炎症因子、凋亡相关蛋白等的表达变化及髓鞘再生情况，明确Etanercept联合NSCs移植治疗的作用机制，从而为今后脊髓继发性损伤的治疗提供新的思路和方法

脊髓损伤后炎症因子的释放尤其是肿瘤坏死因子TNF-α，可引起损伤部位神经元的凋亡，髓鞘的脱失和轴突的破坏。然而细胞移植在治疗脊髓损伤过程中，由于移植NSCs在缺乏外源性营养支持的情况下，移植治疗脊髓损伤的效果较差。目前关于移植NSCs治疗脊髓损伤手段众多，但治疗效果仍有待改进。. 本课题利用慢病毒载体介导TrkC基因修饰NSCs；获得稳定转染的高表达TrkC的NSCs。同时研究制备大鼠脊髓挫伤模型，早期（损伤后约1h）应用 Etanercept(依那西普，阻断TNF-α与TNF受体结合) 腹腔注射，损伤后7天给予TrkC基因修饰NSCs移植和NT-3局部注射，研究Etanercept 联合TrkC基因修饰NSCs 移植治疗脊髓损伤的作用机制。结果：脊髓损伤早期应用Etanercept ，同时移植TrkC基因修饰NSCs和NT-3局部注射能够抑制损伤后的炎症反应造成的继发性脊髓损伤；显著改善脊髓损伤后运动功能的恢复，减小脊髓空洞面积和神经细胞凋亡减少，对脊髓损伤有积极的治疗效果。通过免疫荧光和Westrnblot检测显示, Etanercept 联合TrkC基因修饰NSCs 移植治疗后肿瘤坏死因子TNF-α、凋亡相关蛋白(caspase-3/8/9 和Bax等)的表达下降，组织学染色（HE染色和Nissl染色）检测显示脊髓空洞面积减小及神经元再生增多。该课题为临床治疗脊髓损伤提供了新思路，发现早期应用Etanercept 显著抑制炎症反应引起的继发性损伤，这将具有重要的理论意义和应用前景。