壳聚糖神经导管复合雪旺氏细胞与巨噬细胞膜片修复神经缺损的实验研究

Ideal therapeutic methods for peripheral nerve defect are currently unavailable. Nowadays, nerve autograft is still the most commonly used method in clinical, however, the paucity of donor nerves, the disparity of donor nerve sizes with the recipient sites and the modality mismatch has limited its clinical application. Therefore, it is particularly important to explore an effective alternative autologous nerve transplantation repair of peripheral nerve defect. Recent research indicates that macrophages can induce a polarized vasculature to relieves the hypoxia via vascular endothelial growth factor-a secretion when under the condition of hypoxia. Schwann cells then use the blood vessels as ‘‘tracks’’ to cross the bridge taking regrowing axons with them. Therefore, this project is designed to utilize the feature that chitosan can be used as a good carrier of tissue-engineered nerve and the special advantage of cell sheet in cells - cells and cells - extracellular matrix interaction, through the construction of a cell sheet can be directly transplanted monolayer cell sheet to the parent organization, besides, it also can be stacked into multi-layer cell sheet to abtain tissue of three-dimensional structure and applied in tissue regeneration. This project intends to composite chitosan nerve conduit with schwann cells and macrophages cell sheet to structure a tissue-engineered nerve to repair the nerve defects of rats, and to explore an effective new method to replace autologous nerve grafts.

周围神经缺损目前尚无理想的治疗方法。目前临床最常用的方法为自体神经移植，但常受制于供体神经缺乏，供体神经尺寸与受区的差异及形态不匹配配等。因此，探索能够有效替代自体神经移植修复周围神经缺损的桥接物显得尤为重要。新近研究表明，在缺氧条件下，巨噬细胞通过分泌血管内皮生长因子-a进而诱导产生一个极化的脉管系统来缓解缺氧，之后雪旺氏细胞使用这些血管作为“轨道”穿过神经桥并引导轴突再生。所以，本项目拟利用壳聚糖可作为组织工程化神经的良好载体这一特点，以及细胞膜片所具有特殊的细胞-细胞和细胞-细胞外基质相互作用，通过叠加多层细胞片制得三维结构的组织应用于组织再生的优势，采用壳聚糖神经导管复合雪旺氏细胞与巨噬细胞共培养的细胞膜片共同构建组织工程神经进行修复修复大鼠神经缺损，探索一种能够有效替代自体神经移植的新方法。

周围神经缺损目前尚无理想的治疗方法。目前临床最常用的方法为自体神经移植，但常受制于供体神经缺乏，供体神经尺寸与受区的差异及形态不匹配配等。因此，探索能够有效替代自体神经移植修复周围神经缺损的桥接物显得尤为重要。新近研究表明，在缺氧条件下，巨噬细胞通过分泌血管内皮生长因子-a进而诱导产生一个极化的脉管系统来缓解缺氧，之后雪旺氏细胞使用这些血管作为“轨道”穿过神经桥并引导轴突再生。所以，本项目采用雪旺氏细胞与巨噬细胞共培养后联合移植进行修复修复大鼠神经缺损，探索一种能够有效替代自体神经移植的新方法。通过CCK-8、Transwell、RT-qPCR和Westernblot检测培养至48h的雪旺氏细胞、M2巨噬细胞和M2巨噬细胞-雪旺氏细胞共培养体系的生物学功能，共培养组均高于其他组（p<0.05），建立SD大鼠坐骨神经挤压伤模型，通过He染色、免疫组化观察神经组织以及称重并计算湿重维持率和MASSON观察大鼠双侧腓肠肌差异，实验数据显示共培养组神经修复效果最好。综上本研究确证了M2巨噬细胞对SCs生物学功能的促进作用；M2巨噬细胞和雪旺细胞联合移植能够有希望替代自体神经移植修复周围神经缺损的组织。