低氧诱导bFGF基因修饰神经干细胞对急性脊髓损伤的修复作用及自噬相关的调控机制

To reconstruct the structure and function of nervous tissue by neural stem cell transplantation, treat the spinal cord injury is one of the hot topics in current neuro-regenerative research. However, the survival and differentiation neural stem cell transplantation is not ideal, and using growth factors to promote the differentiation has uncontrollable risks with the regulated level. Basic fibroblast growth factor (bFGF), is one of the necessary neurotrophic factor for the differentiation of stem cells, has great ability in differentiation and proliferation. Our preliminary study found that exogenous bFGF can promoted the recovery of spinal cord injury of rats via regulating autophagy; at the same time, after the transfection of adenovirus vector genetic recombined with bFGF, through genetic recombination under hypoxia, successfully constructed controllable genetic modification of rat embryonic neural stem cells. In this project, the new stem cells above will be transplanted to acute spinal cord injury rats, the expression of bFGF was controlled with hypoxia condition, to explore the influence of controllable genetically modified neural stem cells to the functional recovery of spinal cord injury. And we will observe the changes of the micro environmental factors such as inflammation, oxidative stress, endogenous factors etc.; especially focus on the autophagy related mechanisms in the differentiation of neural stem cells and promotion of the recovery process, and its relationship with bFGF gene regulation. The aim of this project is to provide a novel treatment strategy for spinal cord injury and further deep theory.

利用神经干细胞移植重建神经组织的结构与功能，治疗脊髓损伤是当前神经损伤修复研究的热点。然而，单纯神经干细胞移植存活率、分化水平均不理想，而利用生长因子促分化时又存在量级不可控风险。碱性成纤维细胞生长因子bFGF是干细胞分化必备的神经营养因子之一，具有强大的促分化和增殖能力。本课题组前期研究发现外源性bFGF能够调控自噬促进大鼠脊髓损伤恢复；同时，通过基因重组成功构建低氧诱导的bFGF腺病毒载体，转染后获得可控性基因修饰大鼠胚胎神经干细胞体系。本项目拟将上述新的干细胞体系移植至大鼠急性脊髓损伤模型，通过低氧调控bFGF表达，探讨可调控性基因修饰神经干细胞对脊髓损伤功能恢复的影响，观察炎症反应、氧化应激、内源性因子等微环境因素的变化；重点关注自噬在神经干细胞分化和促恢复过程中的相关机制及其与bFGF基因调控的联系，旨在为脊髓损伤研究提供新的治疗策略和深入的理论基础。

脊髓损伤（spinal cord injury, SCI）是一类破坏性损伤，通常导致患者的功能显著障碍。脊髓损伤的发生过程复杂，包括原发性损伤和继发性损伤。脊髓血管的变化，缺氧，ATP依赖性的丧失和细胞凋亡等在SCI中产生毒性环境，使SCI持续发生。损伤部位产生的胶质瘢痕导致物理障碍的形成，使轴突再生无法通过该区域。利用移植干细胞重塑神经系统的结构功能，是当前治疗SCI的研究热点。然而，单纯神经干细胞移植存活率、分化水平均不理想，而利用生长因子促分化、又存在量级不可控风险。bFGF是干细胞研究中必备的因子之一, 具备强大的促迁移与分化的能力。目前针对SCI的有效治疗策略尚未明确，在临床上非手术和手术治疗未能取得满意的疗效。本课题组前期研究发现外源性bFGF能够调控自噬促进大鼠脊髓损伤恢复；同时，通过基因重组成功构建低氧诱导的bFGF慢病毒载体，转染后获得可控性基因修饰大鼠胚胎神经干细胞体系。本项目拟将上述的干细胞体系移植到大鼠急性脊髓损伤模型中，观察bFGF基因修饰的神经干细胞对脊髓损伤后功能恢复的影响，观察炎症反应、氧化应激、内源性因子等微环境因素的变化；重点关注自噬在神经干细胞分化和促恢复过程中的相关机制及其与bFGF基因调控的联系。本项目成功构建4种基因修饰的原代大鼠胚胎神经干细胞，并分别注入SCI大鼠脊髓损伤中央区。在所有治疗组中，5HRE-bFGF 基因修饰 NSCs组行为学检测实验、组织学研究及神经细胞增殖检测中的治疗效果最佳。同时在本项目的自噬研究基础上，发现基因修饰的干细胞移植治疗和脊髓损伤与自噬存在相互调控关系。本项目基于细胞疗法有促进SCI后功能恢复的潜力，使用干细胞，生长因子和基因载体等三个组合，进一步调控缺氧和SCI微环境，5HRE-bFGF-NSCs移植治疗脊髓损伤已经显示出良好的改善神经功能和功能学恢复，促进NSCs增殖，分化，迁移和轴突再生等，从而抑制SCI引起的自噬，显著促进大鼠SCI的运动行为学功能恢复。为脊髓损伤的研究提供了新的治疗策略和深入的理论基础。