复合细胞因子控释系统的功能化多肽水凝胶支架与神经干细胞联合移植修复脊髓损伤的实验研究

The pathophysiology of spinal cord injury is complicated. The current treatment is difficult to reverse the neurological deficits caused by the cellular level damage. Our previous work found that collagen gel, as a carrier for controlled release of neurotrophic factors, can effectively promote the proliferation and differentiation of neural stem cells. Based on our previous work, we used poly(lactic-co-glycolic acid) (PLGA) microspheres as a carrier containing brain-derived neurotrophic factor (BDNF) to promote neurogenesis and vascular endothelial growth factor (VEGF) to promote angiogenesis. Then, we used collagen gel, incorporated with laminin-derived cell adhesive polypeptides, as a carrier for neural stem cells and PLGA- microspheres to repair of cavity formed in the site of spinal cord injury and promote neurogenesis and angiogenesis. So, we should form a functional polypeptide collagen gel scaffold at first, and then this scaffold containing PLGA-microspheres was cultured with neural stem cells to observe the effects on survival, promotion and differentiation of neural stem cells. Finally, this composite grafts were transplanted to the site of spinal cord injury. This study will provide theoretical basis and experimental support for the clinical spinal cord injury repair treatment.

脊髓损伤的病理生理过程相当复杂，目前尚缺乏有效的治疗手段。我们以往的工作发现胶原凝胶做为神经营养因子控释的载体，可以有效促进神经干细胞的增殖和分化。本课题拟在前期工作的基础上，用聚乳酸聚乙醇酸共聚物（PLGA）制备可控释促血管形成的血管内皮生长因子（VEGF）微球和促干细胞生存、分化的脑源性神经生长因子（BDNF）微球；同时用来源于层粘连蛋白的新型多肽修饰的胶原凝胶作为神经干细胞和细胞因子微球的载体支架，这种新型支架既可以修复脊髓损伤部位形成的空洞，又能为细胞因子和干细胞及脊髓损伤周围组织提供更多的接触机会，从而促进神经血管再生。为此，我们首先将构建功能化多肽胶原凝胶支架，然后复合含有细胞因子的微球并与神经干细胞共培养，观察其对神经干细胞生存、增殖和分化的影响，最后研究该复合移植物对大鼠脊髓损伤的治疗效果，并探讨修复机制。本研究将为修复脊髓损伤开辟新的途径，为临床应用奠定理论基础。

拟采用复合细胞因子控释系统的功能化多肽水凝胶支架与神经干细胞联合移植修复脊髓损伤。本课题通过聚乳酸聚乙醇酸共聚物（PLGA）制备可控释促血管形成的血管内皮生长因子（VEGF）微球和促干细胞生存、分化的脑源性神经生长因子（BDNF）微球；同时用来源于层粘连蛋白的新型多肽修饰的胶原凝胶作为神经干细胞和细胞因子微球的载体支架修复脊髓损伤。首先构建可以携带BDNF和VEGF的PLGA微球，并测量微球的直径，电镜结果显示微球表面光滑，直径分布比较均匀，与胶原凝胶复合良好，ELISA结果显示生长因子在体外可以释放14天，红外光谱、流变测试、溶胀率和导电测试均表明复合生长因子微球的胶原凝胶适合做为移植的支架材料，同时与原代神经干细胞共培养后，通过CCK-8、扫描电镜、LIVE/DEAD染色、免疫荧光和PCR进行检测相关指标，结果显示组织工程化的水凝胶支架可以促进神经干细胞增殖，减少细胞凋亡，并诱导神经干细胞向神经元分化。综上所述，本研究将为修复脊髓损伤开辟新的途径，为临床应用奠定理论基础。项目资助发表SCI论文2篇，核心论文1篇，待发表四篇。培养硕士生2名，2名在读。项目投入经费17万元，支出7.89万元，各项支出基本与预算相符。剩余经费9.11万元，剩余经费计划用于本项目研究的后续支出。