SHH缓释复合纤维蛋白支架促大鼠脊髓损伤修复的研究

In this work, a sonic hedgehog (SHH)-loading composite fibrin scaffold will be constructed. The specific process is as following, firstly, SHH-contained chitosan microcapsules will be prepared with the method of ionotropic gelation and filled into the fibrin scaffold with ectomesenchymal stem cells (EMSCs) as seed cells. The effect of slow-release SHH to the formation of synapse and differentiation of EMSCs into neuron will be investigated in vitro. Then, the SHH-loading composite fibrin scaffold will be planted into the site of injured rat spinal cord. and then to be sacrificed, And the effect on the regeneration and functional recovery of total transected spinal cord of adult rat was analyzed with immunohistochemistry technique, and the tissue reconstruction of the scaffold in the in injured spinal cord will be observed with the electron microscope. At the same time, the rats of each group were estimated with system of the Basso-Beattie-Bresnahan (BBB) locomotion score. This study will provide new idea and lay a reliable experimental foundation for the application of tissue engineering scaffold in spinal cord injury.

本课题选用音猬因子（Sonic hedgehog, SHH）作为细胞分化诱导剂和神经纤维延伸的趋化因子和突触形成因子，制备包封SHH的壳聚糖微胶囊，而后将其梯度填充于纤维蛋白支架，外胚层间充质干细胞（ectomesenchymal stem cells, EMSCs）为种子细胞，构建SHH缓释复合纤维蛋白支架。体外观察该支架缓释释放的SHH对具有神经干细胞特性的EMSCs向成熟神经细胞分化和形成突触的影响。在体外研究的基础上，将该支架移植入大鼠脊髓缺损部位，通过光镜、电镜及免疫组化方法对损伤部位动态观察支架在脊髓内的改建过程,及其对神经再生和突触形成及胶质细胞增生的影响，并对各组大鼠进行Basso Beattie Bresnahan（BBB）运动功能评分，综合评价SHH缓释复合纤维蛋白支架对脊髓损伤后的神经修复作用，进而为组织工程支架移植修复脊髓损伤提供新的研究思路和临床应用提供实验依据。

脊髓损伤的治疗研究已探索多年且取得了重要进展，但目前受损脊髓仍然不能达到结构和功能的完全修复。结合了种子细胞、生物材料及生长因子的组织工程支架，对损伤的组织或器官进行形态、结构和功能的修复与取代，在脊髓损伤修复中具有理想的应用前景。本课题选用音猬因子（Sonic hedgehog, SHH）作为细胞分化诱导剂和神经纤维延伸的趋化因子，用反相微乳法制备包封SHH的壳聚糖缓释微球，而后将其填充于纤维蛋白支架中，外胚层间充质干细胞（ecto-mesenchymal stem cells，EMSCs）为种子细胞，构建含种子细胞且具有缓释功能的组织工程支架，而后用于大鼠脊髓全横断模型的损伤修复。实验结果表明：SHH随着纤维蛋白支架的降解不断作用于损伤部位，诱导EMSCs向神经样细胞和雪旺细胞的分化，促进密集新生神经组织的形成和减少胶质瘢痕的增生，并且有部分的新生神经纤维能够穿越脊髓损伤处胶质瘢痕阻碍，同时，EMSCs在损伤部位存活，增殖分化和迁移，改善了局部微环境，明显促进后肢功能的恢复，该复合纤维蛋白支架有望成为脊髓损伤修复的理想材料。