分子氢改善神经导管修复效能及其机制研究

Peripheral nerve lesions are common injuries in clinical settings with extremely high rates of disability. Schwann cells (SCs) play key roles in promoting nerve regeneration. However, the viability of SCs might be largely affected due to a lack of oxygen in the early stages following injuries and the increased levels of local oxidation products, which leads to suboptimal outcomes in nerve regeneration. Our previous study reported that rats receiving Hydrogen-rich saline achieved better axonal regeneration and functional recovery. Further investigation indicated that Hydrogen was capable of protecting SCs agaist hypoxic damage and up-regulating the secretion of neurotrophic factors, which might be largely associated with its anti-hypoxic and anti-oxidative properties. On the basis of the pioneer studies, the present study focused on establishing a 3D Hydrogen-rich SCs culture system under hypoxia conditions, with the attempt to maintain the viability of SCs and to promote the secretion of neurotrophic factors. Furthermore, a nerve conduit incorporated with Hydrogen-rich gel and SCs was to be developed to bridge a lengthy nerve defect in vivo. In addition, Hydrogen gas inhalation was to be performed postoperatively to exert continuous protection of SCs and to provide a favorable regenerative micro-environment. The axonal regeneration and functional recovery were to be examined by a combination of walking track analysis, electrophysiological assessment, Fluoro-Gold retrograde tracing, as well as morphometric analyses to both regenerated nerves and target muscles. Also, the levels of neurotrophic factors, cell adhesion molecules, myelin-associated molecules, oxidation products, anti-oxidative stress enzymes and apoptosis-related genes were to be evaluated, with the atempt to investigate the mechanism underlying the neuroprotection of Hydrogen. Taken together, the present study was expected to enrich the theory of neural tissue engineering and to raise the feasibility of taking Hydrogen as a neuroprotective agent for peripheral nerve therapies.

周围神经损伤具有高发生率和高致残率，局部缺血缺氧、氧化应激所致雪旺细胞损伤可能是制约神经修复效能的关键因素之一。我们前期报道，分子氢能够促进大鼠自体移植的坐骨神经修复再生和功能恢复；体外实验发现分子氢具有抑制雪旺细胞凋亡及促神经营养因子合成分泌，推测可能与分子氢抗缺氧、抗氧化应激特性密切相关。在此基础上，我们拟：①建立缺氧条件下雪旺细胞培养模型，优化氢保护雪旺细胞参数指标；②构建“富氢凝胶+雪旺细胞”神经导管桥接长节段神经缺损，辅以术后吸入式供氢对雪旺细胞持续保护，改善神经损伤局部再生微环境；③采用组织形态学、神经电生理、行为学及FG逆行示踪等评价神经导管修复效能；④检测神经营养因子、细胞粘附分子、成髓鞘相关分子、氧化应激产物、抗氧化应激酶类及凋亡相关基因表达情况，深入探讨分子氢改善神经导管修复效能分子机制。本项目有望丰富组织工程神经构建理论，为神经损伤修复提供新的思路。

周围神经损伤具有较高的致残率，是最为棘手的临床治疗难题之一。神经组织工程技术的快速发展为神经损伤修复带来新的希望。雪旺细胞在促进神经修复再生过程中发挥着关键作用，亦是构建组织工程神经最为经典的种子细胞。然而，组织工程神经植入早期其内部仍处于缺血缺氧状态，造成雪旺细胞存活率下降；神经损伤局部的氧化应激产物同样对雪旺细胞造成损害。这些因素严重制约组织工程神经的修复效能。因此，保持雪旺细胞的活力在神经修复再生过程中发挥着至关重要的作用。此外，神经导管为周围神经损伤修复提供了良好的再生室，保证神经修复过程中必要的神经营养因子、再生因子促进神经再生。在各种组织工程神经导管中，自体组织瓣制作的神经导管，特别是利用自体骨骼肌的肌外膜制备的神经导管（EMC）表现出明显的优势。本课题组在制备的自体肌外膜导管内部注入基质胶（BD Matrigel），不但可以防止导管塌陷，同时也为神经轴突再生提供良好的通道和再生微环境。我们发现，缺氧状态下富氢培养基有效缓解雪旺细胞凋亡，促进其合成及分泌神经营养因子；富氢液及红景天苷溶液有效改善大鼠坐骨神经修复再生和运动功能恢复水平；基于上述结果构建神经复合导管（EMC-SCs-SDS-Matrigel）并应用于大鼠10 mm坐骨神经缺损的桥接。结果表明，加入SDS-SCs基质凝胶的复合神经导管能有效促进轴突再生和髓鞘化。进一步的研究表明，加入SDS-SCs基质凝胶的神经导管能够提高再生神经运动功能的恢复。行为学分析显示，SDS-SCs基质胶组坐骨功能指数（SFI）明显改善，腓肠肌萎缩部分逆转。免疫荧光分析表明，SCs和小胶质细胞数量增加，有利于神经再生微环境的构建。综上所述，EMC-SCs-SDS-Matrigel复合神经导管可以促进坐骨神经再生和功能恢复的效果，为周围神经损伤的治疗提供了新的途径。