等离子注入改性三维石墨烯促进神经干细胞定向分化和原位生物电信号监测研究

A great variety of mental and brain diseases are increasing largely in modern society and have become one of the major diseases to threaten people health. Neural stem cell (NSC) based therapy provides a promising approach for neural regeneration. For the success of NSCs clinical application, a scaffold is required to provide three-dimensional (3D) cell growth microenvironments and appropriate synergistic cell guidance cues. In this proposal, using 3D graphene as a biocompatible scaffold for NSCs in vitro is attempted and the biological effect of the microstructure is clarified. To further alter the surface topographical, chemical and electrical state of 3 D graphene by plasma ion immersion implantation, the cell behaviors, such as adhesion, proliferation and directional differentiation of NSCs on modified 3D graphene, can be investigated and the intrinsic mechanism could be disclosed. In the process of the directional differentiation of NSCs and the growth of neuronal cells (NCs), the bioelectricity signal could be collected in-situ by high conductive 3D graphene scaffold and an appropriate exogenous electrical stimulation will be feedback to explore the pathways for rapid repair and regeneration of NCs. The purpose of this study is to set up a practical model of modified surface induced the directional differentiation of NSCs by combining of in-situ bioelectricity signal detection and the achievements in this proposal will keep deep insight to understand the cell behavior of NSCs and NCs and provide novel tissue engineer scaffold materials for therapy brain diseases and damage.

各种精神隐疾和重大脑疾病发病率逐年上升，已成为危害人类健康的重大疾病之一。神经干细胞移植是治疗神经系统病变和严重脑损伤的重要途径。采用体外培养，实现神经干细胞的大量增值和分化调控是神经干细胞临床应用的前提。本项目拟制备具有良好生物相容性三维石墨烯，阐明三维石墨烯孔结构大小对细胞生长的影响。利用等离子改性三维石墨烯的表面状态，研究不同表面功能团、润湿性和表面电性等对神经干细胞粘附、增值和分化的影响，阐明石墨烯表面结构和表面化学状态对神经干细胞定向分化和神经细胞生长的影响。结合三维石墨烯的高导电性，原位检测神经干细胞分化和神经细胞成长过程中的生物电信号的变化，并反馈电刺激来研究神经细胞的电活性。研究电流和电压刺激对神经细胞快速修复和再生的作用。结合改性石墨烯表面功能团的作用机理和生物电信号规律，提出神经干细胞定向分化和神经细胞损伤快速修复的途径和方法，为脑疾病和脑损伤的治疗提供新的思路。

基于三维石墨烯及生物材料表面组成和结构对小鼠皮质神经细胞的生物活性和小鼠骨髓间充质干细胞分化进行了研究。主要成果包括：制备了具有高氮含量（7.5%原子比）的三维石墨烯，采用多种方法制备了三维石墨烯结构。利用表面XPS分析和红外等手段研究了离子注入对三维石墨烯表面官能团种类和浸润性的影响。发现O2等离子修饰比NH3等离子修饰的表面更具有浸润性，主要原因由于氧离子注入在表面更易形成羟基，而NH3等离子注入主要形成吡啶和吡咯氮。经过离子注入后，石墨烯表现出明显的细胞毒性，可能由于表面结构被破坏，更容易产生高活性的氧自由基。发现未经修饰的三维石墨烯具有良好的生物相容性，能够促进小鼠皮质神经元的树突棘数量以及轴突的生长。相应的生长蛋白和β-微管蛋白表达稍有增强。基于前面的研究基础，探索了纳米结构和微量离子缓释的表面对骨髓间充质干细胞分化的影响。经对相成骨关基因的检测，发现纳米管表面的成骨和血管生成相关基因表达均有明显上调。并且载有微量元素Mg的纳米管具有明显促进骨髓间充质干细胞向成骨分化和增强成骨细胞增值的功能，因为Mg离子激活了ERK1/2信号通路。本研究结果为生物基材表面功能化设计用于神经元和骨髓间充质干细胞的相关研究提供了新思路。