三维培养下物理微环境对神经干细胞自我更新与定向分化调控规律及机理研究

Self-renewal and directed differentiation of neural stem cells (NSCs) are regulated by the joint physical and biochemical factors in the niche. Currently, most of the studies at home and abroad focus on manipulation of NSC behaviors by biological and chemical cues at the two-dimensional (2D) level. There is a huge gap between these 2D studies and the real physiological situation of NSCs, which are located in the central nervous system with three-dimensional (3D) structure. Also, these studies ignore the importance of the physical cue, including electrical field in the central nervous system, in regulating NSC behaviors, which are quite rare in the current literatures. Because of the complexity of the in vivo studies and the difficulty to control, 3D culturing can mimic the physiological environment and bring great convenience to the study. This project intends to adopt the most advanced material preparation techniques to fabricate 3D culture system for NSCs. By taking advantages of the spatial structure and excellent electrical conductivity, we can build the physical niche of NSCs in a controllable fashion. Under this condition, we will the regulatory effects of physical cues in the 3D system, including topology and electric field stimulation, on the NSC self-renewal and directed differentiation. Also, we will summarize the differences between 3D culture and 3D culture and clarify the possible mechanisms. Furthermore, the structures and functions of neurons and neural network will be validated in the 3D culturing system. This study is expected to compensate for the lack of understanding the regulatory role of physical factors in stem cell research in the 3D space, and provide a theoretical basis for neural stem cell-based tissue engineering and regenerative medicine.

神经干细胞的自我更新与定向分化受其微环境中的物理及生化因子的联合调控。目前国内外研究多聚焦在二维平面上研究生化因子对神经干细胞行为的调控，这与神经干细胞所处的中枢神经系统是一个立体的三维体系有较大的差别，且忽略了神经系统中包括电场因素等在内的物理因子的重要性，其对神经干细胞行为调控的研究也鲜见于报道。由于体内研究的复杂性和不可控性，神经干细胞体外三维培养可尽量模拟生理环境并为研究带来极大便利。本项目拟采用最前沿的材料制备技术，制备干细胞的三维培养支架，利用材料的空间拓扑结构与优异的导电性，可控构建神经干细胞的物理微环境，研究此三维微环境中拓扑结构与电场刺激等对神经干细胞自我更新与定向分化的调控规律，归纳其与二维培养的异同及机制，并进一步地验证三维培养下分化的神经元及神经网络的结构功能。本研究有望弥补三维物理因子对干细胞调控研究的不足，为基于神经干细胞的组织工程和再生医学提供理论依据。

神经干细胞（neural stem cells, NSC）更新与分化受其微环境中的物理及生化因子的联合调控，目前国内外研究多聚焦在二维平面上研究生化因子对NSC行为的调控，这与NSC真实的生理环境有较大的差别，且忽略了神经系统中包括电场因素等在内的物理因子的重要性。在三维环境下研究物理因子对NSC行为调控具有重要的科学意义及一定的应用价值。本项目在资助期内，采用了先进的材料制备技术，制备了多种基于石墨烯材料的干细胞的三维培养支架，并且利用材料表面的拓扑结构与优异的导电性，可控构建了NSC的物理微环境，以二维培养为对照，系统研究了此三维微环境中拓扑结构与电场刺激等物理因子对NSC自我更新与定向分化的调控规律，并进一步地阐明了子代细胞结构与功能的改变情况，包括神经元的生长发育、神经网络的结构与功能等。具体的研究成果包括：（1）建立了多种基于石墨烯材料的适宜干细胞生长、调控其增殖与分化的三维培养体系；（2）系统地研究了石墨烯的体外及体内生物安全性研究；（3）研究了生物材料物化性质对干细胞及神经元行为的调控规律及作用机制；（4）以人工耳蜗及石墨烯材料，制备了声电转化细胞培养装置，归纳了人工耳蜗声电刺激对神经元及神经干细胞行为的调控规律及可能的机制。以上结果对于理解NSC及子代细胞在三维培养下的行为规律及物理因子对干细胞结构及功能调控提供了实验证据，研究成果也弥补了本领域内相关研究的不足，为基于神经干细胞的组织工程和再生医学也提供了理论依据。本项目资助期内，共培养硕士研究生3名，博士研究生2名。参加国内外学术会议4次，发表6篇SCI文章，包括Advanced Materials，Biomaterials等高水平杂志。