纤维蛋白胶支架搭载外胚间充质源雪旺细胞移植修复大鼠脊髓损伤

Transplantation of fibrin scaffolds containing ectomesenchymal Schwann cells to repair the spinal cord injury in rats

目的:观察外胚层间充质源的雪旺细胞在损伤脊髓中的迁移情况、组织相容性,评价纤维蛋白支架搭载该细胞移植治疗急性脊髓损伤的疗效。方法:(1)成年大鼠鼻中隔活体取出部分鼻黏膜制成细胞悬液,自然纯化并扩增鼻黏膜外胚层间充质干细胞(ectomesenchymal stem cells,EMSCs),用雪旺细胞诱导培养基将其诱导为雪旺细胞并用红色荧光进行标记。(2)建立脊髓针刺损伤模型并移植标记细胞,术后10 d,观察雪旺细胞在组织内的存活和迁移情况,并评价其组织相容性。(3)健康SD大鼠60只,制作脊髓横断模型,随机分为3组:单纯脊髓横断(SCI)组、纤维蛋白胶(Fibrin)移植组和细胞-支架(F-C)移植组,术后每周进行运动行为学评分。术后12周取出脊髓组织,采用免疫组织化学染色和Western Blot方法检测脊髓横断部位神经纤维重建和髓鞘再生情况,以及神经丝蛋白(NF-200)、生长相关蛋白-43(GAP-43)和髓鞘碱性蛋白(MBP)的表达变化。结果:(1)EMSCs来源的雪旺细胞体外分化扩增良好,免疫荧光结果显示其高表达S100、GFAP、P75、CNpase等表面标志蛋白。(2)移植进入体内的雪旺细胞存活良好并发生一定距离的迁移,且与脊髓内神经纤维有良好的相容性。(3)术后2周开始直至12周,F-C组较SCI组大鼠后肢运动功能有较明显恢复,差异有显著意义(P<0.05);Fibrin组亦有一定程度的恢复,但程度较轻;SCI组几乎没有恢复迹象。免疫组化染色结果显示:SCI组断端未见神经轴索通过,Fibrin组可见少量神经纤维通过,F-C组可见多量神经网络状结构。Western Blot检测结果显示:F-C组神经丝蛋白(NF-200)、生长相关蛋白-43(GAP-43)和髓鞘碱性蛋白(MBP)相对含量均高于Fibrin组和SCI组(P<0.05)。结论:外胚层间充质源雪旺细胞移植对于脊髓损伤后的组织学和功能学恢复有明显促进作用,纤维蛋白做为移植修复脊髓损伤的生物支架具有良好的应用前景。

Objective： To study the migration and histocompatibility of the ectomesenchymal Schwann cells in the injured spinal cord, and observe the curative effect of the transplanting the ceils to repair the injured spinal cord. Methods ： （ 1 ） The EMSCs derived from adult rat nasal respiratory mucosa were culture and then induced to differentiation into Schwann cells （SCs） by the SCs culture medium. The labelled ectomesenchymal Schwann cells with red fluorescent were tranplanted into injured spinal cord of rats. （2） The migration and histocompatibility of the SCs were observed after transplanting these cells into the needle damage spinal cord model for 10 days. （3）60 Sprague-Dawley host rats were randomly assigned into three groups ：SCI group （laminectomy and transection of spinal cords） ;fibrin group （fibrin was transplanted immediately after SCI）, and the fibrin cell （F-C） group （fibrin scaffolds containing ectomesenchymal SCs were trans- planted after SCI）. The rats of each group were respectively estimated by the BBB locomotor scores every week after operation. The injured spinal cord segments with or without the scaffolds were removed out and examed in the 12th week after operation. The proliferation regeneration of the nerve fibers and the reconstruction of the myelin sheath were analysed with immunohistochemistry and immunoblotting metheods. Results ： （ 1 ） Ectomesenchymal Schwann cells showed well differen- tiation and amplification in vitro. It indicated a high level of expression when immunofluorescence-stained for S100, GFAP, P75 and CNpase. （2）The labelled ectomesenchymal Schwann cells that transplanted into injured spinal cord showed well migration and histocompatibility. （3） The rats of the F-C group showed significant improvement in BBB scores by week 2 that persisted through week 12 when compared to the rats of the SCI group （P 〈 0.05 ） ;A certain degree of improvement was showed in the rats of fibrin group ; The rats of the SCI group showed almost no change. The result of the Im- munohistochemistry indicated that there was almost no nerve fiber between the lacerated ends of SCI group. The result also showed there was a small quantity of nerve fibers among the lacerated ends of fibrin group but a network of nerve fibers be- tween the lacerated ends of the F-C groups. Western Blotting results showed the expression NF200, GAP43 and Myelin basic protein（MBP） in F-C group was the strongest among all the three groups, followed by that in the Fibrin group and the SCI group. Statistical analysis showed that the expression levels of all three markers were significantly different be- tween the FC group and the fibrin group （ P 〈 0.05 ）. Conclusion： The fibrin scaffolds containing ectomesenchymal Schwann ceils could improve the behavioral and histological recovery after SCI significantly; Fibrin as a biomaterial has a bright perspective in construction of the tissue engineering scaffold to repair the injured spinal cord.