【作者】 马军；

【导师】 崔福斋；

【作者基本信息】 清华大学 ， 材料科学与工程， 2006， 博士

【摘要】 神经元网络被认为是人体内最复杂的组织形式之一,实现神经网络损伤后的修复与重建以及体外模拟构建神经网络,是神经组织工程和神经芯片等领域的重要研究目标,其中,一个基础问题就是理解和控制神经元与材料的界面反应。本论文以化学腐蚀的硅基片,仿生制备的静电自组装多层薄膜,微接触印刷图案作为材料表面修饰设计的界面模型,进行皮层和海马神经元等原代细胞培养,并运用激光共聚焦扫描显微镜和原子力显微镜等材料科学表征手段,观察细胞和材料的界面反应变化,以此来研究材料的表面特征与神经细胞相互作用的规律和诱导形成神经元网络的机理。上述神经细胞在模型界面上的培养结果表明,神经细胞和这些优化设计的模型界面都能够达到良好的相容性和亲和性,其中,神经元在纳米粗糙度的硅基片(Ra=20-70nm)和透明质酸基的多层薄膜上都可以粘附生长,并且能够形成成熟致密的神经元网络结构。通过原子力显微镜对神经细胞与硅的界面结构研究发现,这个界面是由分级结构组装而成,神经细胞通过改变自身周围的微环境来提高存活率,促进生长发育。在多层薄膜的结构中发现薄膜层数和最外层组分对轴突的发育具有一定的影响,并且不同类型的神经细胞对多层薄膜的组分有不同的喜好倾向。使用微接触印刷的生物大分子图案可以实现图案化神经元网络的构建。通过比较不同基团和不同大分子“墨水”,发现在羟基化的玻璃片上印刷的PEI图案能够比较有效的控制神经元的粘附和突起的生长。在免疫组化的结果中,PEI图案上布满了密集排列的纤维样的神经元突起,原子力显微镜观察到这些纤维结构之间还存在相互的联系。进一步的观察中还看到了神经元胞体附近的突触结构,这是图案化网络中的神经元之间通过轴突和树突建立了相互联系的重要证据。此外,为了便于神经细胞和材料相互作用的研究,发展了一种使用电位敏感荧光探针,结合激光共聚焦扫描显微镜,测量神经细胞膜电位和电场分布的方法,可以实现细胞膜电位分布的三维重构。更多还原

【Abstract】 Neuronal network is considered as one of the most complex tissues of human beings. The subject of the interface between neurons and the surface of biomaterials has of late been receiving much attention in the research fields of nerve tissue engineering and neuron chip because it has implications for repair and recruitment of the neuronal networks in vivo and in vitro. Chemically etched silicon substrates, polyelectrolyte multi-layered films by electrostatic self-assembly and patterns of biological macromolecules, were proposed as the model surfaces to study the interfacial reactions with the primary neural cells such as cortical and hippocampal neurons. The interfacial structures between cells and materials, induced effects to neural cell growth and network development by materials were studied in details using the confocal and atomic force microscopy.From the culturing results of the primary neural cells, the cells show good viability and compatibility with the designed model surfaces, including the silicon wafers with nano-scale roughness (Ra=20-70nm), hyaluronic acid based multilayered films. The neural cells can adhere and grow on the substrates, form mature dense neural networks. From the atomic force microscopic results, the interface between the neurons and silicon substrates were constructed by hierarchical structures which promoted the cell adherence and development. In addition, the neural cells were considered to change the surrounded microenvironments to improve their viability and living conditions. Meanwhile, on the multilayered films which mimicked the extracelllular matrices, the neurite outgrowth was influenced by the polycationic outmost layer and bi-layer number to certain extent. In addition, it seemed that different neurons like different polycations. The hippocampal neurons prefer poly (allylamine hydrochloride) while collagen attracts cortical neurons. Patterned neural network was fabricated using the microcontact printing of biological macromolecules. From the comparisons of different surface functional groups and different“ink”, polyethylene (PEI) pattered on hydroxyl modified glass slides have achived the best performance among the tested combinations. From the immunohistochemical results, filament-like neurites runs in parallel on the PEI patterned grids. These dense neurites have relations between each other from the atomic force microscopic results. Furthermore, we found synaptic structures near the neuron body, which is the evidence of connection between axons and dendrites.In addition, a protocol of measuring membrane potential and electric field of neurons in vitro by confocal microscopy was proposed to evaluate the living status of neural cells, which was suggested to have potential applications in the study of interaction between the neural cells and materials. This is the first report of a reliable method for three-dimensional visualization of potential voltage of neurons at the best of our knowledge.更多还原