具有促血管神经再生功能的透明质酸复合载体携带嗅鞘细胞修复大鼠脊髓损伤的研究

Spinal cord injury (SCI) often resulted in patients with different degree of paralysis. But so far, it is lack of effective treatment. The application of biomaterials seeded with olfactory ensheathing cells (OECs) is a very good approach. However, the effect is restricted, since the biomaterials can not creat a proper niche for neural restoration. Our previous studies mainly focused on the application of grafted hyaluronic acid (HA) based directional channel scaffold modified with Nogo receptor antibody (antiNgR) in repairing of SCI in rats, this method can promote the regeneration of nerve fibers, but the quantity is small and failed to bridge into damage zone. The aim of this project is to improve the scaffold by adding several angiogenic molecules to promote angiogenesis, reconstruct a better local niche for neural restoration and to explore the effects and mechanisms of implantation of the newly composite scaffold seeded with OECs into the lesion area of rat spinal cord. In brief, the HA scaffold is constructed with longitudinally multi-channel. Afterwards, it will be modified with antiNgR to block inhibitors (Nogo, MAG, OMgp) and mixed further with poly(lactic-co-glycolic acid) (PLGA) microspheres containing vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for promoting local angiogenesis. The new scaffold will sustained release two kinds of angiogenic factors and antiNgR. Bioactivity of this HA composite scaffold will be assessed by neurons, OECs and vascular endothelial cells culture in vitro. Then, the composite scaffold seeded with OECs will be transplanted into the injured area of rat model with T9-10 dorsal hemisection. The changes of angiogenesis, microcirculation reconstruction, nerve fiber regeneration, OECs survival and migration, synaptic connections will be examined in grafting region with electronmicroscope, immunofluorescence histochemistry including anterograde dextran tracing. The functional recovery of the rats will also be evaluated with BBB scale, CatWalk gait analyse. Hope that our study can provide a new approach to restore SCI by promoting local re-vascularization and rehabilitation of the spinal cord. Our study will also provide the experimental basis for clinical application on SCI.

脊髓损伤常造成患者不同程度的瘫痪，目前尚缺乏有效治疗手段。应用生物材料携带嗅鞘细胞的综合疗法显示出优势，但因未能建立良好的局部微环境而影响疗效。我们前期应用接枝NgR抗体的透明质酸定向通道支架修复大鼠脊髓损伤，促进了神经再生，但纤维数量少且未能跨越损伤区。本课题拟在此基础上，进一步对材料进行改性并携带嗅鞘细胞对大鼠脊髓损伤进行实验性治疗。即在具有定向通道的透明质酸-NgR抗体复合物支架中，添加包封血管内皮生长因子和促血管生成素-1的聚乳酸聚乙醇酸共聚物微球，形成可缓释两种血管生成因子和NgR抗体的透明质酸水凝胶载体。将此载体携带嗅鞘细胞植入大鼠脊髓损伤区，观察移植物和周边组织血管再生、微循环重建、神经纤维再生以及植入的嗅鞘细胞存活迁移及通路重建状况，并对动物的行为进行评估。希望通过本研究能提供一个更好地促进脊髓损伤局部血管和神经再生、组织修复及功能恢复的新方法，为临床应用奠定实验基础。

脊髓损伤常造成患者不同程度的瘫痪，目前尚缺乏有效治疗手段。应用生物材料携带嗅鞘细胞（OECs）的综合疗法显示出优势，但因未能建立良好的局部微环境而影响疗效。我们前期应用接枝NgR抗体的透明质酸（HA）定向通道支架修复大鼠脊髓损伤，促进了神经再生，但纤维数量少且未能跨越损伤区。本研究在此基础上，进一步对材料进行改性并携带OECs对大鼠脊髓损伤进行实验性治疗。即在具有定向通道的HA-NgR抗体复合物支架中，添加包封血管内皮生长因子（VEGF）和促血管生成素-1（Ang1）的聚乳酸聚乙醇酸共聚物（PLGA）微球，形成可缓释两种血管生成因子和NgR抗体的HA水凝胶载体。将此载体携带OECs植入大鼠脊髓损伤区，观察移植物和周边组织血管、神经纤维再生以及植入的OECs存活迁移情况，并对动物的行为进行评估。结果显示，构建的HA支架具有纵向多通道结构，通道之间借孔隙相通；PLGA微球可释放VEGF和Ang1；HA水凝胶可促进人脐动脉内皮细胞、OECs在材料上粘附、生长；将携带OECs的HA复合支架植入大鼠脊髓损伤区后，材料与脊髓组织整合良好，有大量神经细胞迁移进入支架内部；植入4周后发现，HA支架植入可显著减轻胶质瘢痕形成，减小损伤面积；植入8周、14周后，免疫荧光染色、天青-美兰染色及透射电镜观察可见，在HA复合支架植入组，移植区材料内部有大量新生的血管和有髓神经纤维，并发现有典型的突触结构；行为学测评结果显示，植入HA复合支架可明显改善术后大鼠的运动功能。结果表明，具有定向通道结构、接枝NgR抗体并携带PLGA微球和OECs的HA复合支架植入脊髓损伤区后可诱导神经细胞向支架材料内迁移，抑制胶质瘢痕形成，促进血管生成和神经纤维再生，改善大鼠运动功能。具有定向通道结构的HA复合支架携带OECs植入脊髓损伤区可有效改善损伤局部微环境，促进大鼠脊髓损伤后组织修复和运动功能恢复。

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