SHH缓释纤维蛋白支架移植促大鼠脊髓损伤的修复

SHH slow-release fibrin scaffold promotes function recovery of the spinal cord injury in rats

目的:研究应用可缓释音猬因子(sonic hedgehog,SHH)的纤维蛋白胶(fibrin glue,FG)支架促大鼠脊髓完全横断损伤的修复效果。方法:(1)制作体外缓释SHH的纤维蛋白胶支架模型,探究缓释效果。(2)选用健康的SD大鼠60只,制作大鼠脊髓完全横断模型,随机分为三组:单纯脊髓横断组(SCI),纤维蛋白胶组(FG),音猬因子-支架移植组(F-SHH)。术后每周记录大鼠双后肢功能活动(BBB评分)。术后12周取出脊髓组织,采用免疫组织化学染色和免疫蛋白印迹检测,观察脊髓损伤的近远端神经元存活情况及神经丝蛋白(neurofilament200,NF200),生长再生相关蛋白(growth associated protein-43,GAP43),胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)表达情况。结果:(1)载SHH的纤维蛋白胶具有良好的缓释SHH的效果;(2)F-SHH组BBB评分从术后到第十二周较FG、SCI组上升明显(P<0.05);(3)免疫组化结果显示:F-SHH组脊髓横断端有一些神经元存活且排列规则,许多神经纤维未完全溃变。SCI组细胞凋亡明显,纤维溃变严重,退变空泡大,FG组较SCI组稍好;(4)Western Blot结果显示:F-SHH组NF200,GAP43相对含量高于FG、SCI组(P<0.05),而GFAP相对含量低于FG、SCI组(P<0.05)。结论:SHH缓释纤维蛋白支架缓释效果良好,对大鼠脊髓完全横断损伤的修复有明显的促进作用,SHH对于脊髓损伤的修复机理值得进一步研究。

Objective：To investigate the transplantation of fibrin glue scaffolds that can release sonic hedgehog （SHH） slowly to promote the repair of spinal cord after compete transection injury in rats.Methods：（1）The model of slow released SHH fibrin glue was made in vitro to explore the release performance of scaffold.（2）60 Sprague-Dawley rats were randomly assigned into there groups：SCI group（laminectomy and transection of spinal cord）;FG group（fibrin glue was transplanted immediately after SCI）;F-SHH group（fibrin scaffolds containing sonic hedgehog were transplanted after SCI）.The rats of each group were respectively estimated by the BBB locomotor scores every week after operation.The injured spinal cord segments of each group were got out and examined in the 12th week after operation.The proliferation regeneration of the nerve fibers and the survival of the neurons were analysed with immunohistochemistry and immunoblotting metheods.Results：（1） Fibrin scaffolds containing SHH showed good effect of slow-releasing SHH.（2） The rats of F-SHH group showed significant improvement in BBB scores compared to the rats of SCI and FG group.（3） The result of immunohistochemistry indicated that some neurons survived and arranged evenly,many of which were lacerated ends of F-SHH group.The SCI group showed significant cell apoptosis,serious fiber degeneration,and large vacuolar degeneration.The FG group was better than SCI.（4） Western Blotting results indicated the expression of NF200 and GAP43 in F-SHH group was the strongest among all the three groups,followed by that in FG group and SCI group.The result of the GFAP expression was reversed.Statistical analysis showed the expression levels of all three markers were significantly different between the FG group and SCI group（P 〈 0.05）.Conclusion：Fibrin glue containing SHH has good effect of slow-release.SHH slow-release fibrin scaffold could improve the behavioral and histological recovery of spinal cord injury.The machanism of SHH for repair gspinal injure crod is worthy of further study.