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背景:神经干细胞是一类具有自我更新和多向分化潜能的特殊细胞,是研究神经系统的发生、发育、发展规律及其内在的分子生物学、细胞学调控机制的理想载体,在神经系统疾病及损伤的治疗中具有广阔的应用前景。探讨一套较为简便、有效和实用的神经干细胞体外培养体系是其应用的前提和基础。目的:观察血清培养基并辅以碱性成纤维细胞生长因子对小鼠神经干细胞体外培养的影响。设计:单一样本观察。单位:右江民族医学院组织学与胚胎学教研室。材料:2个月龄昆明种小白鼠5只,雌雄不拘。方法:实验于2003-06/2005-05在广西医科大学组织学与胚胎学试验室完成。①采用贴壁培养法,从成年小鼠大脑皮层分离并体外培养成年小鼠大脑皮层细胞,制成细胞悬液后将其置入DMEM/F12(1∶1)液(包括体积分数为0.15的胎牛血清,碱性成纤维细胞生长因子20μg/L,青霉素100u/mL和链霉素100u/mL)培养,获得细胞克隆。②应用免疫组织化学技术检测克隆细胞巢蛋白的表达。主要观察指标:①培养细胞形态学观察。②培养细胞苏木精-伊红染色观察结果。③神经干细胞鉴定。结果:①培养细胞形态学观察:细胞接种后,绝大多数细胞在12h内附壁,细胞变扁平,接种后第3天,细胞开始生长,细胞数量增多,接种后第5天时生长成为数10个细胞到数百个细胞克隆,到第七八天,细胞占据瓶底80%～90%。经传代后仍成黏附生长,细胞形态规则,边界清楚。②培养细胞苏木精-伊红染色观察结果:可见细胞圆形或椭圆形,胞质少呈嗜酸性,细胞核大而圆,单个居中,着色深。③神经干细胞鉴定:免疫荧光检测显示,细胞巢蛋白抗原呈阳性。结论:分离的小鼠大脑皮层细胞在血清培养基并辅以碱性成纤维细胞生长因子的培养条件下,具有很强的增殖能力,传代后表达巢蛋白,依然具有神经干细胞的特征。
BACKGROUND: Neural stem cells are a kind of special cells with the potential of self-renewal and multidirectional differentiation. They are ideal carriers for studying the genesis, development and development of nervous system and its intrinsic molecular biology and cytological regulation mechanism. In neural system diseases And injury treatment has broad application prospects. To explore a set of more simple, effective and practical system of neural stem cells in vitro culture system is the premise and basis for its application. Objective: To observe the effect of serum medium supplemented with basic fibroblast growth factor on the culture of mouse neural stem cells in vitro. Design: Single sample observation. Unit: Youjiang Nationalities College of Histology and Embryology. Material: 2 months old Kunming mice 5, male or female. Methods: The experiment was performed at the Laboratory of Histology and Embryology, Guangxi Medical University from June 2003 to May 2005. ① Adherent culture method was used to separate and culture adult mouse cerebral cortex cells from adult mouse cortex. After the cell suspension was made into DMEM / F12 (1: 1) solution (including the volume fraction of 0.15 Fetal bovine serum, basic fibroblast growth factor 20 μg / L, penicillin 100 u / mL and streptomycin 100 u / mL) to obtain a cell clone. ② Immunocytochemistry was used to detect the expression of nestin in cloned cells. MAIN OUTCOME MEASURES: ① cultured cells morphological observation. ② cultured cells hematoxylin-eosin staining results. ③ neural stem cell identification. Results: (1) Morphological observation of cultured cells: After inoculation of cells, the majority of cells adhered to the wall within 12h, and the cells became flattened. On the third day after inoculation, the cells started to grow and the number of cells increased. On the fifth day after inoculation, the number grew to 10 A cell to hundreds of cell clones, to the seventh eight days, the cells occupy 80% to 90% of the bottom. After passage still become adhesion growth, cell morphology rules, the border is clear. ② cultured cells hematoxylin - eosin staining results: visible cells were round or oval, less eosinophilic cytoplasm, large and round nucleus, single center, deep color. ③ neural stem cell identification: immunofluorescence test showed that cells were positive for nestin antigen. CONCLUSION: Isolated mouse cerebral cortex cells have strong proliferative ability under the condition of serum culture medium supplemented with basic fibroblast growth factor. Nestin is expressed after passage and still has the characteristics of neural stem cells.