神经干细胞复合顺序释放生长因子的胶原海绵修复脊髓损伤

There is no effective treatment for neurological deficits resulting from spinal cord injury (SCI) despite significant medical and surgical advances. The combined strategy with neural stem cells (NSCs) and scaffolds offers a promising approach to promote SCI recovery. Creating a permissive environment in vivo to improve neural stem cell survival and neural differentiation is becoming increasingly attractive. Epidermal growth factor (EGF) is known to promote proliferation of NSCs in vitro culture, as well as in vivo environments. Brain-derived neurotrophic factor (BDNF) is of particular interest with regards to directing cell fate toward neuronal specification, and promoting neuronal survival, synaptogenesis, and axonal regeneration. However, the simultaneous use of EGF and BDNF can not achieve an excellent synergistic effect. In traditional culture system, NSCs were expanded and maintained in an undifferentiated state in medium supplemented with EGF, after a certain time, the medium was changed with BDNF to induce neuronal differentiation. However, it is not suitable for in vivo implantation. We propose here a strategy based on sequential delivery of EGF and BDNF from collagen sponges to promote NSC survival and proliferation, and then neuronal differentiation and axon regeneration. In order to achieve the sequential delivery, we used here the genetically engineered BDNF that was fused with a collagen-binding domain (CBD) derived from collagenase. In the prior study, we have fabricated CBD-BDNF successfully, and it was found that CBD-BDNF could bind to collagen specifically and a prolonged release was achieved. We thus hypothesize that in the system of collagen sponges loaded with EGF and CBD-BDNF, an initial release of EGF followed by a slow and sustained release of CBD-BDNF could be achieved. Growth factor release rate will be monitored over time in vitro, the in vitro study of NSCs will be conducted to test the effect of sequential delivery of EGF and CBD-BDNF on NSC proliferation and differentiation, and furthermore, the combined therapy of NSCs and collagen sponges incorporated with sequential release of EGF and BDNF for extreme transected rat SCI model will be evaluated. The present study will contribute to a better design of new scaffolds with growth factor sequential therapy and a better understanding of NSC regulation, and provide valuable information for clinical use of NSCs.

脊髓损伤修复是临床医学重大难题。神经干细胞复合支架提供了新途径，促进其存活、增殖和向神经元分化是焦点问题。表皮细胞生长因子(EGF)可促进神经干细胞增殖，脑源性神经营养因子(BDNF)可促进其向神经元分化、轴突再生，但二者同时使用效果不理想，传统培养通常顺序使用含EGF和BDNF的培养基，但不适于体内植入，本项目拟建立支架担载EGF、BDNF顺序释放体系，期望促进神经干细胞先存活、增殖，再向神经元分化、轴突再生。为了实现顺序释放，改造生长因子，将从胶原酶中筛选的与胶原结合的功能短肽(CBD)与BDNF融合，课题组前期已证实CBD-BDNF与胶原涂层结合实现了延缓释放。研究胶原海绵支架担载EGF和CBD-BDNF顺序释放动力学，对神经干细胞增殖、分化的作用，以及神经干细胞复合此支架对大鼠脊髓全横断损伤修复效果。通过本研究可望建立顺序释放新方法，加深对神经干细胞调控的认识，为临床应用基定基础。

神经干细胞移植进行脊髓损伤修复的关键问题是如何改善微环境，促进其增殖和向神经元细胞分化。经过本项目三年的实施，我们系统地研究了胶原海绵支架的功能化修饰，其对神经干细胞增殖与定向分化的调控规律，及神经干细胞复合胶原海绵进行脊髓损伤修复的效果。首先制备了多孔结构胶原海绵支架，同时采用基因工程的方法制备了具有胶原特异结合短肽的脑源性神经营养因子（CBD-BDNF）。进一步对胶原海绵支架进行功能化修饰，分别从添加神经营养因子和拮抗抑制分子两个角度进行研究：①通过具有胶原特异结合区的神经营养因子建立了表皮生长因子（EGF）、CBD-BDNF顺序释放体系；②通过化学交联建立了EGFR抗体西妥昔缓慢释放体系。更重要的是，我们研究了功能化胶原海绵支架对神经干细胞增殖与定向分化的调控规律。结果表明EGF、CBD-BDNF顺序释放体系可促进神经干细胞增殖，向神经元细胞分化；EGFR抗体西妥昔缓慢释放体系可缓解髓鞘蛋白的抑制作用，促进神经干细胞向神经元分化，抑制其向星形胶质细胞分化。进一步，我们研究了神经干细胞复合功能化胶原海绵支架对大鼠脊髓半横断损伤的修复效果，发现EGFR抗体西妥昔修饰的胶原海绵支架在体内可促进移植的神经干细胞向神经元分化，促进神经丝和突触的形成，最终促进大鼠运动功能的恢复。此外，在原研究内容基础上拓展，研究了梯度功能支架的制备及其对神经干细胞的调控作用，发现CBD-基质细胞衍生因子-1 (Stormal cell-derived factor-1, SDF-1α) 浓度梯度支架可诱导神经干细胞定向迁移。依托本项目，我们已发表4篇学术论文，研究成果对于神经干细胞微环境调控、神经干细胞定向分化调控、以及脊髓损伤修复具有一定的指导意义。