大鼠嗅球神经干细胞增殖及诱导分化的实验研究

发布时间：2018-03-30 09:53

本文选题：大鼠　切入点：神经干细胞　出处：《郑州大学》2006年硕士论文

【摘要】：目的：建立新生大鼠嗅球神经干细胞(neural stem cells，NSCs)体外分离培养方法。通过对不同发育阶段大鼠嗅球NSCs体外培养的形态学观察及免疫荧光化学染色结果分析，探讨不同发育阶段大鼠嗅球NSCs增殖、分化特性。使用不同浓度的胎牛血清、IGF-I和BDNF诱导大鼠嗅球NSCs的分化，探讨促进大鼠嗅球NSCs向神经元分化的因素，为大鼠嗅球NSCs的深入研究与临床应用打基础。材料与方法：从新生大鼠嗅球组织中分离NSCs，采用神经细胞球形成法，以无血清培养液，使用生长因子(bFGF、EGF)，进行体外扩增培养，以机械分离法连续传代，相差显微镜及电子显微镜观察细胞形态，免疫细胞化学法检测Nestin表达，四甲基偶氮唑盐(MTT)比色法和流式细胞仪检测大鼠嗅球NSCs的增殖能力。取胎龄18天(embryonic day 18，ED18)、出生24h以内(postnatal within 24h，P0)及2月龄(2 month，M2)的大鼠嗅球组织行单细胞克隆，免疫荧光化学染色鉴定嗅球NSCs，，并计数分化为神经元、星形胶质细胞和少突胶质细胞的比例。用免疫细胞化学染色和流式细胞仪分选细胞法观察不同浓度的胎牛血清、IGF-I和(或)BDNF对嗅球NSCs分化为神经元、星型胶质细胞、少突胶质细胞的影响。结果：光镜下原代培养见细胞呈大小不一、悬浮生长的细胞团，边界清晰，折光性好，细胞团中细胞排列较为致密，传代培养的细胞形态特点与原代培
[Abstract]:Objective: to establish a method of isolation and culture of neural stem cells from olfactory bulb neural stem cells (NSCs) of newborn rats in vitro. The morphological observation and immunofluorescence staining results of NSCs in olfactory bulb of rats at different stages of development were observed. To investigate the proliferation and differentiation characteristics of olfactory bulb NSCs in rats at different developmental stages, the differentiation of olfactory bulb NSCs was induced by different concentrations of fetal bovine serum IGF-I and BDNF, and the factors that promoted the differentiation of rat olfactory bulb NSCs into neurons were discussed. To lay a foundation for the further study and clinical application of rat olfactory bulb NSCs. Materials and methods: NSCs were isolated from the olfactory bulb of newborn rats. NSCs were isolated from the olfactory bulb tissue of newborn rats. The cells were cultured in vitro using serum-free medium and growth factor BFGFG EGFN. The cells were subcultured by mechanical separation method. Phase contrast microscope and electron microscope were used to observe cell morphology and immunocytochemistry method was used to detect the expression of Nestin. The proliferative ability of rat olfactory bulb NSCs was detected by MTT colorimetry and flow cytometry. Single cell clones were performed in the olfactory bulb tissues of rats with embryonic day 18 (ED18), postnatal within 24 h (P0) and 2-month old (2 monththophane M2) at gestational age of 18 days. NSCs of olfactory bulb were identified by immunofluorescence staining and differentiated into neurons. The ratio of astrocytes to oligodendrocytes. Immunocytochemical staining and flow cytometry were used to observe the differentiation of NSCs from olfactory bulb to neurons and astrocytes in fetal bovine serum of different concentrations. The effect of oligodendrocytes. Results: under the light microscope, the cells in primary culture were different in size and size. The cell clusters with suspending growth had clear boundary, good refraction and compact arrangement of cells in the cell clusters. The morphological characteristics and primary culture of the cells in passage culture were similar to those in the primary culture.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

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