Anosmin-1蛋白联合嗅鞘细胞抑制胶质瘢痕形成并促进脊髓损伤修复的机制研究

Reactive astrogliosis following spinal cord injury (SCI) forms a glial scar barrier that prevents the penetration of regenerated axons through the lesion site. Olfactory enstheathing cells (OECs) reduce reactive astrocyte stress response via FGF signalling, however, they exhibit inadequate capability of inhibiting glial scar formation. Extracellular matrix protein anosmin-1 is essential to promote regenerated olfactory axons to infiltrate the glial barrier on the outmost surface of olfactory bulb via FGF signalling pathway. Such characteristics suggest that anosmin-1 as combined with OECs could help regenerated axons to penetrate through glial scar barrier following SCI, therefore implicating its potential application in SCI treatment. In a rat SCI model, we will inject anosmin-1 alone or combined with OECs into rat SCI lesion site to observe regenerative axonal penetration through astroglial barrier and recovery of forelimb function. Expression of FGF signalling components and reactive astrogliosis will be analyzed by immunohistochemistry, immunofluorescency and Western blot. To investigate whether anosmin-1 assists cellular interactions of OECs with astrocytes in vitro, we will use recombinant anosmin-1, MAPK and PI3K inhibitors on OECs and astrocytes co-cultures, which are pre-treated with heparin or desulfated heparins to induce astrocyte stress response. This study will provide evidence for anosmin-1 combined with OECs in inhibiting glial scar formation and then helping the regenerative axons to penetrate through glial scar barrier after SCI via regulation on FGF signalling pathway, therefore establishing novel molecular basis for anosmin-1 as a potential molecular intervention for SCI treatment.

脊髓损伤后的星形胶质细胞应激反应，形成阻止再生神经穿透的胶质瘢痕。嗅鞘细胞虽可通过FGF通路减轻星形胶质细胞应激反应，但不能充分抑制胶质瘢痕形成。申请者前期研究证实，Anosmin-1蛋白调节FGF通路，促进再生嗅神经穿透嗅球表面的胶质层, 因而成为辅助嗅鞘细胞抑制胶质瘢痕形成、促进再生轴突穿透胶质屏障的重要候选因子。本课题设想通过anosmin-1 联合嗅鞘细胞，以期解决脊髓损伤后胶质瘢痕形成这一难题。我们拟应用单侧皮质脊髓束损伤的大鼠，经anosmin-1蛋白联合嗅鞘细胞移植，观察前爪定向伸展功能的恢复、胶质瘢痕形成、再生轴突瘢痕穿透和FGF信号复合物的表达。体外共培养嗅鞘细胞与星形胶质细胞, 经anosmin-1、FGF通路抑制剂处理后，观察星形胶质细胞应激反应的变化。本研究将论证anosmin-1联合嗅鞘细胞治疗脊髓损伤及其与FGF通路相关性，为治疗脊髓损伤提供新的研究思路及方法。

源于嗅粘膜的嗅鞘细胞是一种特殊的胶质细胞，通过形成细胞通道来辅助嗅神经的轴突穿透嗅球表面胶质层。嗅鞘细胞的分化程度决定了其特定的生物功能，即成熟的嗅鞘细胞帮助嗅神经轴突迁移，而未成熟的嗅鞘细胞决定了嗅神经穿透嗅球表面的胶质层，这一特性使得嗅鞘细胞移植可以帮助损伤的脊髓神经穿过星形胶质细胞形成的疤痕层，用于治疗脊髓损伤。Anosmin-1是一种糖基化的细胞外基质粘附蛋白, 在嗅球表面表达，可能通过FGF信号系统来调节嗅鞘细胞的分化成熟程度，从而决定着嗅球的发育。我们对鸡胚胎通过siRNA技术、嗅球移植块和原代嗅球细胞培养的实验，多个层面证实了anosmin-1蛋白通过FGF信号系统来调控嗅鞘细胞的分化和成熟程度，保持嗅鞘细胞处于嗅球发育早期的未成熟状态，使嗅鞘细胞在与星形胶质细胞相互作用的过程中，保持胶质层处于开放状态，便于嗅神经穿透嗅球表面的胶质层。该研究成果为anosmin-1 蛋白参与嗅鞘细胞和星形胶质细胞界面的形成以及嗅鞘细胞促进脊髓损伤修复提供了坚实的实验依据和分子生物机理的支持。Anosmin-1 蛋白通过FGF信号系统调控嗅鞘细胞分化成熟的作用机理，已经发表于Eur J Neurosci。我们接着又对anosmin-1蛋白与肝素特定点位的硫基结合来调控FGFR1/FGF2/肝素信号复合体形成的分子机理进行研究, 通过应用ELISA、等离子共振顺序注射和细胞信号通路的实验，发现anosmin-1蛋白可以将经典FGF信号通路依赖肝素6-O硫基的特性转变为主要需要肝素2-O和N-点位上的硫基，这一改变需要肝素的长度大于18个六碳糖基，这一结果为anosmin-1蛋白通过肝素特定硫基调控FGF通路提供了新的机理，并为FGF信号通路活性的调控建立了新的分子作用模式。anosmin-1蛋白可能通过调节肝素2-O和N-点位上的硫基的特性，来调节嗅鞘细胞以及星形胶质细胞相互作用，调控特定组织和部位的神经细胞以及嗅觉部位的嗅鞘细胞中FGF通路的活性，从而发挥促进神经再生的作用，并为anosmin-1蛋白用于脊髓损伤修复提供了分子生物机理方面的支持。