壳聚糖导管复合人羊膜间充质干细胞来源的雪旺氏细胞与温敏型胶原水凝胶修复神经缺损的实验研究

Peripheral nerve injury is a common and frequently-occurring disease in clinical. Autologous nerve transplantation is still the "gold standard" of repairing the peripheral nerve defect, however, the paucity of donor nerves, the disparity of donor nerve sizes with the recipient sites and the complications of donor sites have limited its clinical application. Therefore, it is particularly important to explore an effective alternative autologous nerve transplantation repair of peripheral nerve defect. Schwann cells play a crucial role in peripheral nerve regeneration, but it is hard to extract a large number of purified schwann cells in a short time in clinical. Our previous study has demonstrated that human amniotic membrane mesenchymal stem cells can be induced to differentiate into schwann cells in vitro, and when the induced cells were transplanted to denervated flap on SD rats, obvious nerve regeneration can be detected. Therefore，on the basis of confirming human amniotic mesenchymal stem cells can be induced to differentiate into schwann cells firstly, to combine the feature that chitosan can be used as a good carrier of tissue-engineered nerve and the superiority that the strong cell adhesion force, the favourable water and nutrient exchange characteristics of temperature sensitive collagen hydrogels, putting the induced schwann cells as seed cells, combine with temperature sensitive collagen hydrogels filling chitosan nerve conduits to construct tissue engineering artificial nerve to repair sciatic nerve defect on rat. The project aims to explore an effective alternative autologous nerve transplantation new method for repairing peripheral nerve defect.

周围神经损伤是临床常见病、多发病。自体神经移植仍是目前修复周围神经缺损的“金标准”，但常受制于供体神经缺乏、供体神经尺寸与受区的差异及供区并发症等。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。雪旺氏细胞在周围神经再生中起到至关重要的作用，但临床上很难在短时间内获取大量纯化的雪旺氏细胞。前期研究发现，人羊膜间充质干细胞可在体外诱导分化为雪旺氏细胞，将其移植于SD大鼠失神经支配皮瓣，可检测到明显的神经再生。所以，本项目首先在明确人羊膜间充质干细胞可诱导分化为雪旺氏细胞的基础上，结合壳聚糖可作为组织工程化神经的良好载体这一特点及温敏型胶原水凝胶具有细胞粘附力强，水分、养分交换特性良好等优点，以诱导的雪旺氏细胞作为种子细胞，联合温敏型胶原水凝胶注射填充壳聚糖神经导管构建组织工程人工神经修复大鼠坐骨神经缺损，旨在探索一种能够有效替代自体神经移植修复周围神经缺损的新方法。

周围神经损伤后神经再生及其功能修复的机制尚不明确。本研究通过将人羊膜间充质干细胞定向诱导分化为雪旺氏细胞样细胞，并通过免疫荧光检测、qPCR和Western blot检测诱导前后细胞内S-100、P75、GFAP的mRNA和蛋白表达情况；通过ELISA检测诱导过程中上清液中BDNF、NGF、NT3含量。本研究表明，hAMSCs具备体外诱导诱导分化为SCLCs的潜能，可作为修复周围神经损伤的SCs的种子细胞；实验过程中发现hAMSCs诱导成SCLCs可能受某种miRNA调控，进一步研究发现其机制可能是miR-146a-3p可调控hAMSCs诱导成雪旺细胞。在大鼠坐骨神经损伤模型中，足迹测定法及坐骨神经功能指数评价结果提示SCLCs移植组神经再生及神经功能恢复能力均优于其他实验组。进一步实验表明，miR-214通过靶向调节c-Jun促进SCLCs的迁移和髓鞘形成，而在SCLCs中过表达miR-214可以进一步促进周围神经再生和坐骨神经损伤的功能恢复。该研究为周围神经损伤的治疗提供了新的依据和方向。目前，已发表本基金支持的论文5篇。