新型HA/RGDP/GO神经支架的制备及其联合hUMSCs在脊髓损伤神经修复中的作用研究

Spinal cord injury (SCI) is a disease with high mobility and mortality, for which there is no effective treatment in clinic, yet our finding that human umbilical cord-derived mesenchymal stem cells (hUMSCs) can promote the neural repair of nerve injury brings new hope to SCI. The prior problem to be resolved is to promote the efficiency of hUMSCs' proliferation and neural differentiation. Recently, it has been discovered that neural scaffold plays an important role in improving the stem cells' proliferation and neural differentiation efficiency. However, the regulatory effect of neural scaffold on hUMSCs is still unclear enough. Herein, we will use hyaluronic acid (HA), polypeptide RGDP and conductive graphene oxide (GO) to fabricate HA/RGDP/GO composite hydrogel as the neural scaffold, and study the regulatory effect of neural scaffold on hUMSCs' proliferation, neural differentiation and neural repair effect. FTIR, SEM and rheometer will be used to characterize the physical and chemical property of hydrogel, optimize the preparation technology. Immunohistochemical staining, Western Blot (WB) and RT-PCR will be utilized to study the relationship between HA/RGDP/GO hydrogel' component and cellular proliferation and neural differentiation. Animal models of SCI will be prepared, and histology and BBB behavioristics will be used to evaluate the neural repair ability of neural scaffold and hUMSCs. The main aims of research are to bulid novel neural scaffold, improve the neural repair effect, and provide scientific bases for the transformation and application of stem cells and the cure of SCI.

脊髓损伤（SCI）致残率死亡率高，临床上无特效疗法。人脐带间充质干细胞（hUMSCs）移植能促进损伤后的神经修复，为SCI治疗带来新思路。移植后细胞增殖及神经分化效率低的问题急需解决。近年发现神经支架在促进干细胞增殖及神经分化中发挥重要作用，但对hUMSCs的调节作用，目前还不够清楚。因此，本项目拟采用透明质酸（HA）、多肽RGDP及氧化石墨烯（GO）制备HA/RGDP/GO水凝胶神经支架，研究其对hUMSCs的增殖、神经分化及神经修复作用的影响。利用FTIR、SEM、流变仪等仪器表征水凝胶理化性能，优化制备工艺。采用免疫组化、WB、RT-PCR等技术研究水凝胶组份与细胞增殖及神经分化的关系。制备SCI动物模型，利用组织学和BBB行为学等方法评价神经支架联合hUMSCs诱导神经修复的能力。本项目旨在构建新型神经支架，增强hUMSCs的神经修复作用，为干细胞转化应用及SCI治疗提供科学依据。

脊髓损伤（spinal cord injury, SCI）是一种具有高死亡率和致残性的神经损伤性疾病，可导致损伤部位及以下永久性的感觉丧失和运动功能障碍，给患者造成了巨大的心理压力和家庭负担，临床上至今无特效治疗方法。SCI 主要分为原发性和继发性损伤，组织撕裂和血管结构破坏后出现原发性损伤，损伤后若不及时控制，炎性细胞浸润、组织水肿、氧自由基形成等会引发更为严重的继发性损伤，最终导致神经元坏死并抑制轴突再生。大部分的病变是由继发性损伤所致，所以SCI 治疗的核心问题是减轻继发性损伤，使神经元得以修复。间充质干细胞是一种具有高度增殖和分化能力的多潜能干细胞，间充质干细胞的移植为 SCI 的修复研究提供了一种有效策略。. 人脐带间充质干细胞（human umbilical cord mesenchymal stem cells, hUC-MSCs）由于取材方便、免疫原性低、分化潜能高、与胚胎干细胞相比涉及的伦理问题少等，广泛应用于干细胞移植治疗。尽管干细胞移植在 SCI 治疗方面展示出了巨大的潜力，但移植后干细胞在病灶部位滞留率、存活率及分化效率低等问题依然制约着干细胞治疗效果，这些问题亟待解决。. 大量研究表明水凝胶支架材料可以提高干细胞移植的治疗效果，因此构建具有良好生物相容性、可降解及可注射的水凝胶支架材料有利于基于干细胞移植的 SCI 治疗。明胶三维水凝胶支架与细胞外基质（extracellular matrix, ECM）具有相似的特性，包括高营养渗透性、高含水量和可调节的机械性能，这些共同为细胞提供了良好生存微环境。可注射型水凝胶在植入过程中的侵入性较小、能填充不规则的缺损，为受损脊髓搭建连接的桥梁，这在组织修复和再生领域具有巨大的应用潜能。但明胶水凝胶在体内稳定性较差极易被胶原酶酶解，文献报道利用对羟基苯丙酸（hydroxybenzene propanoic acid, HPA）接枝明胶后可提高其稳定性。本研究利用半乳糖氧化酶（galactose oxidase, GalOx）和辣根过氧化物酶（horseradish peroxidase, HRP）催化的方法共价交联接枝苯酚基的明胶聚合物形成水凝胶。双酶交联水凝胶能促进 hUC-MSCs 在体外的存活、增殖及神经分化，此外，该水凝胶负载 hUC-MSCs 可显著提高 SCI 小鼠的神经功能修复。