基于软弹性GelMA水凝胶的三维力学微环境构建及其调控神经干细胞向神经元分化研究

Treatment of central nervous system (CNS) injuries or disorders using neural stem cell (NSC) transplantation often suffers from low neuronal differentiation, leading to insufficient nerve loop reconstruction and signal propagation, thus greatly limiting its therapeutic effect. Studies have shown that this is closely related to the inappropriate mechanical properties of the extracellular microenvironment, which is an important determinant of NSC behaviors including neuronal differentiation. However, conventional studies are mainly limited to 2-dimensional (2D) mechanical microenvironment, making it difficult to obtain results comparable with those of 3D in vivo microenvironment. Additionally, the matrix materials used in existing studies to construct 3D mechanical microenvironment are usually stiff and brittle, and thus may not be able to simulate the natural soft and elastic microenvironment of NSCs. Therefore, the aim of this project is to construct a 3D mechanical microenvironment which can mimic the natural microenvironment of NSCs and to regulate their neuronal differentiation, with the ultimate goal of achieving a functional neural tissue. To achieve this goal, in this study, a new type of soft and elastic hydrogel, photocrosslinkable gelatin, will first be synthesized to construct a microstructure which can model in vivo 3D microenvironment of NSCs. The effect of mechanical microenvironment (e.g., varying matrix stiffness and applying tension) on neuronal differentiation of NSCs and functions of generated neural tissues will then be investigated. The findings of this project will open new insights about how to direct NSC fate by tuning the 3D mechanical microenvironment and reveal the underlying mechanism. The results will also provide important experimental data and theoretical reference for treatment of CNS injuries and disorders.

神经干细胞（NSC）移植是临床治疗中枢神经系统（CNS）损伤最具前景的方法之一。但是，移植后NSC定向分化为功能性神经元较少，难以重建受损神经环路，严重影响其疗效。既往研究表明，力学微环境对NSC向神经元方向分化有重要影响。然而，相关研究多限于二维环境，且所用基质材料硬度较高且弹性较弱，难以模拟在体NSC所处柔软且有弹性的三维微环境。目前，三维力学微环境如基质刚度和应力/应变对NSC向神经元分化的影响尚不明确。因此，本项目拟首先合成新型软弹性光交联明胶（seGelMA），构建能模拟NSC自然生长的三维力学微环境，进而改变其基质刚度和拉伸应变，调控NSC向神经元分化，从而实现神经电传导功能体外重建。本项目将从“力-行为-功能”三方面揭示三维力学微环境诱导NSC向神经元分化的规律和机制，为体外构建可调控NSC行为和功能的三维微环境提供理论和实验依据，为临床NSC移植治疗CNS损伤提供重要思路。

前期研究结果表明细胞外基质的力学性能可以调控神经细胞的行为和功能，如细胞形态、粘附和增殖、以及其相关基因和蛋白表达，从而诱导神经组织再生和功能修复。因此，研究材料刚度如何影响神经细胞的生长和功能变化，在神经修复的临床应用中起着关键作用。本研究在构建不同弹性模量水凝胶的基础上，研究了其力学性能对神经细胞生长与分化的影响。进一步，从基因和蛋白表达水平上，阐释了水凝胶力学性能调控神经细胞行为与分化机理。研究结果表明，水凝胶机械强度可以通过调节背根神经节细胞中Ntn4，Sema3D和Epha4基因的表达，来控制神经细胞的生长。为了进一步模拟体内神经细胞生长的三维（3D）微环境，我们制备了一系列具有不同机械性能（1至110 kPa）的水凝胶。这些水凝胶可支持神经细胞Neuro-2a（N2a）的生长，因而可用于深入探究3D机械刺激对神经细胞分化的影响。在3D封装神经细胞后，我们将载细胞的三维水凝胶拉伸至125％和150％，以研究在不同程度拉伸度下神经细胞的分化。研究结果显示，该3D水凝胶具有良好的生物相容性。免疫荧光结果表明，适当的机械刺激有助于神经细胞的分化。此外，在拉伸水凝胶中，N2a细胞有分化为星形胶质细胞的趋势。qPCR结果进一步证明，拉伸后水凝胶中N2a细胞的GFAP和β-III tubulin表达显着增加，而Nestin的表达显着降低，这表明机械刺激可促进神经干细胞分化为神经细胞和神经胶质细胞。总之，本项目从“模量-细胞-基因-信号途径”研究角度出发，揭示了材料基质刚度与神经细胞行为之间的关系，为神经移植物的设计和制备提供了坚实的理论基础。