音猬因子缓释聚多巴胺纤维蛋白支架促进大鼠脊髓损伤的修复

Sonic hedgehog-polydopamine-fibrin scaffold promotes recovery of spinal cord injury in rats

背景:近来多数研究将组织工程材料与干细胞或因子复合来改善动物脊髓损伤模型损伤处的微环境,以增加作用时长、提高恢复效果与预后。目的:观察音猬因子-聚多巴胺-纤维蛋白支架修复大鼠脊髓损伤的效果。方法:采用真空冷冻干燥机制作纤维蛋白胶,将其置入盐酸多巴胺溶液中浸泡24 h进行交联,再将交联后的支架置入音猬因子溶液中吸附交联24 h,制备音猬因子-聚多巴胺-纤维蛋白支架。取60只雌性SD大鼠建立脊髓损伤模型,随机分4组处理:A组不植入任何材料,B组植入纤维蛋白支架,C组植入聚多巴胺-纤维蛋白支架,D组植入音猬因子-聚多巴胺-纤维蛋白支架,术后12周内进行下肢运动功能BBB评分;术后12周取脊髓损伤处组织,分别进行组织学观察(苏木精-伊红与免疫组化)与Western blot检测。实验方案经江苏大学动物实验伦理委员会批准。结果与结论:①从术后2周开始各组大鼠下肢运动功能开始恢复,D组术后5-12周的BBB评分高于其他3组(P<0.05),D组下肢运动功能恢复最好;②苏木精-伊红染色显示,C、D组可见新生的神经纤维组织,C组新生神经纤维组织的量与密度低于D组;③免疫组织化学染色显示,D组横断处可以观察到大量线性排列的新生神经纤维,髓磷脂碱性蛋白、生长相关蛋白和神经丝蛋白阳性率高于其他3组,胶质纤维酸性蛋白阳性率低于其他3组;④Western blot检测显示,D组髓磷脂碱性蛋白、生长相关蛋白和神经丝蛋白高于其他3组(P<0.05),胶质纤维酸性蛋白表达低于其他3组(P<0.05);⑤结果表明,音猬因子-聚多巴胺-纤维蛋白具有良好的缓释性能,对大鼠脊髓损伤修复有明显促进作用。

BACKGROUND: Recently, most studies have combined tissue engineering materials with stem cells or factors to improve the microenvironment of animal models of spinal cord injury to increase the duration of action, improve the recovery effect and prognosis. OBJECTIVE: To investigate the effect of sonic hedgehog-polydopamine-fibrin scaffold on the repair of spinal cord injury in rats. METHODS: Fibrin glue was made using a vacuum freeze-dryer. The prepared fibrin glue was immersed in a dopamine hydrochloride solution for 24 hours for cross-linking. Then the cross-linked scaffold was placed in a factor solution for adsorption and cross-linking for 24 hours. Sonic hedgehog-polydopamine-fibrin scaffolds were prepared. Sixty female SD rat models of spinal cord injury were established and then divided into four groups: In the group A, no material was implanted. In the groups B, C and D, fibrin scaffolds, polydopamine-fibrin scaffolds, and sonic hedgehog-polydopamine-fibrin scaffolds were implanted respectively. The Basso, Beattie and Bresnahan(BBB) locomotor scale score of lower limb locomotor function was evaluated within 12 weeks after surgery. At 12 weeks post-surgery, the tissue at the site of spinal cord injury was collected for histological observation(hematoxylin-eosin and immunohistochemical staining) and western blot assay. This study was approved by the Animal Ethics Committee of Jiangsu University, China. RESULTS AND CONCLUSION:(1) From 2 weeks after surgery, the lower limb locomotor function of rats in each group began to recovery. At 5-12 weeks after surgery, the BBB score of group D was significantly higher than that of the other three groups(P < 0.05). Rats in group D had the best recovery of locomotor function of the lower limb.(2) Hematoxylin-eosin staining revealed newly generated nerve fibers in the groups C and D, and that the number of density of new nerve fibers in group C was lower than that in group D.(3) Immunohistochemical staining showed that a large amount of linearly arranged new nerve fibers were observed in the completely transected site of rat spinal cord. In group D, myelin basic protein-, growth related protein-and neurofilament protein-positive rates were significantly higher(P < 0.05), and glial fibrillary acidic protein-positive rate was significantly lower, compared with the other three groups.(4) Western blot assay revealed that in group D, the protein expression of myelin basic protein, growth related protein and neurofilament protein was significantly higher(P < 0.05), and the protein expression of glial fibrillary acidic protein was significantly lower(P < 0.05), compared with the other three groups.(5) These results suggest that sonic hedgehog-polydopamine-fibrin has a good sustained-release performance, which can greatly promote the repair of spinal cord injury in rats.