纤维蛋白原(Fg)与纤维蛋白(Fb)差异性调控EMSCs命运的机制及其在脊髓损伤修复中的应用

Fibrin (Fb) gel can enhance the repair of spinal cord injury (SCI). Previous studies found that Fb could promote the differentiation of embryonic stem cells into neurons. However, we found that fibrinogen (Fg), the soluble precursor of Fb, could de-differentiate ectomesenchymal stem cells (EMSCs). Yet, the molecular mechanisms are to be clarified. Based on previous reports and our preliminary experiments, we hypothesize that Fg and Fb may regulate EMSCs’ fate via integrin and YAP1 which can be linked by 14-3-3/Lats1 complexes. Therefore, in order to explore the underlying mechanism, we will analyze the impact of Fg or Fb on the expression levels of integrin and YAP1, the nuclear translocation of YAP1, and the changes of the expression of Oct-4 and Sox-2 in vitro, which will be followed by the analysis of the interaction between integrin and 14-3-3/Lats1 complexes. Finally, in vivo experiments will be used to confirm the hypothesis and to explore the promotion of SCI repair by applying the mechanism of opposite regulation of stem cells’ fates by Fg and Fb. This project will clarify the mechanism of differential regulation of EMSCs’ fate by Fg and Fb and provide a new strategy for SCI therapy.

纤维蛋白胶(Fb)可促进脊髓损伤修复。研究表明Fb能促使胚胎干细胞分化为神经元，而我们发现其前体纤维蛋白原(Fg)可诱导外胚间充质干细胞(EMSCs)逆分化。然而，Fg与Fb差异性调控干细胞命运的机制尚不清楚。基于文献和预研结果，我们推测：Fg与Fb可能通过整合素及其效应分子YAP1调控EMSCs命运，且14-3-3/Lats1复合物是沟通整合素和YAP1的关键。因此，本项目拟通过体外实验分析Fg和Fb对整合素、YAP1表达水平及YAP1核转位的影响，以及Oct-4、Sox-2表达的变化，探明Fg和Fb差异性调控EMSCs命运的机制；并分析整合素与14-3-3/Lats1相互作用的情况；最后通过体内实验进一步验证调控机制，并将组织工程支架调控干细胞命运应用于脊髓损伤修复。研究结果将阐明Fg与Fb差异性调控EMSCs命运的机制，为组织工程支架/干细胞移植修复脊髓损伤提供新理念。

干细胞组织工程支架移植修复脊髓损伤具有广阔的应用前景。然而，支架与干细胞之间的相互作用，特别是支架对干细胞命运的调控研究却尚未受重视。本课题发现分子组成类似而性状不同的游离纤维蛋白原(Fg)和凝胶状纤维蛋白(Fb)对鼻粘膜来源的外胚间充质干细胞（EMSCs）的命运存在差异性调控。由此我们深入研究了此现象的内在机制。体内外实验的结果表明： Fg与Fb对EMSCs命运的差异性调控伴随着Integrin/14-3-3/YAP1的表达或分布的改变；单独干扰Integrin αV和14-3-3 epsilon的表达并不影响Fg与Fb差异性调控EMSCs命运，推测可能与Integrin和14-3-3的其它亚型的代偿效应有关； Integrin内吞引起的自噬在Fg诱导EMSCs逆分化过程中起着重要作用，若抑制自噬则Fg引起EMSCs死亡；低剂量的蛋白酶体抑制剂可促进Fg诱导EMSCs逆分化，故可联用MG-132与Fg诱导EMSCs逆分化；在EMSCs内Integrin、14-3-3及Lats1存在相互作用；直接将Fg注入脊髓损伤部位会阻碍脊髓组织修复及功能恢复；将Fg逆分化EMSCs所获得的iNSCs与EMSCs共同负载于Fb上移植入脊髓损伤部位能显著促进脊髓损伤修复。. 本课题揭示了不同性状的支架可调节干细胞内信号转导从而调控细胞命运，通过更精确地调控干细胞命运可促进脊髓损伤修复。特别地，本项目揭示了一种新的组织工程支架构建方案——将逆分化的自体干细胞与定向自体干细胞相互结合，互相补充，构成功能更健全的人造组织。