海马神经元之间的大分子通讯

发布时间：2018-03-19 17:39

本文选题：海马神经元　切入点：原子力显微技术(AFM)　出处：《中国人民解放军军事医学科学院》2010年硕士论文　论文类型：学位论文

【摘要】： 目的：利用原子力显微镜技术(AFM)和转基因技术研究原代培养的海马神经元表面超微结构、相互间的连接结构及其相关功能。材料和方法：取新生12小时以内的C57小乳鼠,在无菌条件下原代培养海马神经元细胞。选择生长良好的海马神经元用2.5%的戊二醛(生理盐水配制)固定30min,然后用流动三蒸水轻轻冲洗样品,以去除样品表面的盐结晶。剪掉培养皿的皿壁,用502胶水固定于载玻片上,待其在空气中自然干燥后,置于AFM的扫描器上,进行扫描成像,成像模式为大气环境下的接触模式(Contact Mode)。分别取新生48小时的转基因RFP C57乳鼠、EGFP C57乳鼠(其脑部特异性转入绿色荧光蛋白和红色荧光蛋白)各一只,在无菌条件下原代混合培养海马神经元细胞。选择生长良好的海马神经元在荧光显微镜下观察并拍照。结果：本实验中培养的海马神经元生长状态良好。(1)利用AFM观察显示：正常海马神经元表面光滑,起伏均匀、规律,神经元多为单个生长,突起之间交织成网,相互间有突触形成。同时,我们还观察到海马神经元间存在膜性连接以及长程非突触性突起连接结构；(2)利用荧光显微镜观察显示：在共培养的细胞中,发现两个通过膜性连接的海马神经元中的一个细胞内,同时存在红色荧光蛋白和绿色荧、光蛋白。结论：我们在以往的工作中利用AFM观察到海马神经元的一些利用其它显微技术难以发现的超微结构,还发现了一些用突触结构难以解释的连接结构,并对其中之一的膜性连接结构进行了初步的研究：(1)再次证明原子力显微技术是研究细胞表面超微结构的有用工具；(2)为神经元间的膜性和长程非突触性突起连接提供了更直接的证据；(3)发现了神经元之间的膜性连接结构可能存在大分子的物质的交换或者转运。由此推测,神经元之间的膜性连接结构可能不仅存在于同一动物的生命发展初期,还有可能在后天一定的刺激条件下形成,以起到信息传递甚至物质转运的作用。
[Abstract]:Objective: to study the surface ultrastructure, connection structure and related functions of hippocampal neurons cultured in primary culture by AFM and transgenic techniques. Materials and methods: C57 pups were collected from newborn mice within 12 hours. Hippocampal neuronal cells were cultured in aseptic condition. Hippocampal neurons with good growth were fixed with 2.5% glutaraldehyde (normal saline) for 30 min. To remove salt crystallization from the surface of the sample, cut off the dish wall of the petri dish, fix it on the slide with 502 glue, and after it is naturally dried in the air, put it on the AFM scanner for scanning imaging. The imaging model was contact mode in the atmospheric environment. One newborn RFP C57 newborn mouse (with brain specific transfer to green fluorescent protein and one red fluorescent protein) was taken from the newborn RFP C57 newborn mouse, which was transformed into green fluorescent protein (GFP) and red fluorescent protein (GFP C57), respectively, in which the brain was transferred specifically to green fluorescent protein (GFP) and red fluorescent protein. Hippocampal neuronal cells were cultured in aseptic condition. Hippocampal neurons with good growth were selected to be observed and photographed under fluorescence microscope. Results: the cultured hippocampal neurons in this experiment were in good growth state. The results showed that the surface of normal hippocampal neurons was smooth. The ups and downs are uniform, regular, neurons are mostly single growth, processes interweave into a network, and synapses form among each other. At the same time, We also observed membranous junctions between hippocampal neurons and long range nonsynaptic neuronal junctions. Using fluorescence microscopy, we observed that in co-cultured cells, Red fluorescent protein and green fluorescence were found in one of two hippocampal neurons via membranous junctions. Conclusion: in our previous work, we have used AFM to observe some ultrastructures of hippocampal neurons that are difficult to find by other microscopic techniques, as well as junctional structures that are difficult to explain by synaptic structures. A preliminary study of the membranous junctional structure of one of them (1) proves that atomic force microscopy (AFM) is a useful tool for studying the ultrastructure of the cell surface, and that atomic force microscopy (AFM) is a useful tool for studying the ultrastructure of the cell surface. This provides more direct evidence of the possible exchange or transport of macromolecular substances in the membranous junction structure between neurons. The membranous junction structure between neurons may exist not only in the early stage of life development of the same animal, but also in the postnatal stimulation, which may play the role of information transmission and even material transport.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R33

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