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背景:对于神经干细胞的分离培养,目前多采用胰蛋白酶对组织细胞予以消化,但消化时间较难把握。目的:采用胰酶消化与机械分离法相结合的方式对昆明种小鼠胚胎脑神经干细胞进行分离、培养,并进行初步的免疫组织化学检测。设计、时间及地点:细胞学体外观察,于2006-10/2007-09在广西医科大学基础医学实验室完成。材料:孕14~16d的昆明种小鼠由广西医科大学实验动物中心提供。方法:分离昆明种小鼠胎鼠的脑组织,经胰酶消化加机械吹打后,在加入碱性成纤维细胞生长因子和表皮细胞生长因子的B27无血清DMEM/F12培养基中培养。主要观察指标:用免疫细胞化学方法鉴定分离的神经干细胞。结果:培养24h后,细胞以悬浮方式生长,聚集成团;48h后形成由数十个细胞组成的细胞球,形态规则,体积大小不等,细胞无突起,形成典型的神经球,可传代扩增。免疫细胞化学染色结果示细胞巢蛋白呈阳性表达。结论:在含有表皮细胞生长因子、碱性成纤维细胞生长因子的无血清B27培养基条件下,有利于小鼠胚胎神经干细胞的体外培养和传代增殖。
Background: For the isolation and culture of neural stem cells, trypsin is used to digest tissue cells, but the digestion time is more difficult to grasp. OBJECTIVE: To isolate and culture mouse embryonic neural stem cells from Kunming mice by means of trypsin digestion and mechanical separation, and to perform preliminary immunohistochemical detection. DESIGN, TIME AND SETTING: The cytology in vitro observation was performed at the Basic Medical Laboratory of Guangxi Medical University from October 2006 to September 2007. MATERIALS: Kunming mice of pregnant 14 ~ 16d were provided by Experimental Animal Center of Guangxi Medical University. Methods: The brain tissue of Kunming mice was isolated and digested with trypsin and mechanical beating, then cultured in B27 serum-free DMEM / F12 medium supplemented with basic fibroblast growth factor and epidermal growth factor. MAIN OUTCOME MEASURES: Isolated neural stem cells were identified by immunocytochemistry. Results: After culturing for 24 h, the cells grew in suspension and aggregated into clusters. After 48 h, cells formed by dozens of cells were regular in shape and size. The cells did not protrude to form typical neurospheres, increase. Immunocytochemical staining showed positive expression of nestin. CONCLUSION: Under the condition of serum-free B27 medium containing epidermal growth factor and basic fibroblast growth factor, it is in favor of in vitro culture and passage proliferation of mouse embryonic neural stem cells.