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背景:体外培养获得神经干细胞的有效方法是干细胞实验研究中很值得关注的问题。目的:观察应用含有不同生长因子的培养基体外培养神经干细胞的有效方法。设计:单一样本观察。单位:四川大学生命科学院细胞生物学实验室。材料:孕14dSD大鼠10只,DMEM/F121∶1,碱性成纤维细胞生长因子,表皮生长因子;巢蛋白抗体IgG,β-微管蛋白Ⅲ抗体IgG,胶质纤维酸性蛋白抗体IgG,生物素标记二抗和三抗、异硫氰酸荧光素标记二抗。方法:实验于1999/2001在四川大学生命科学院细胞生物学实验室完成。①从胎鼠前脑取出脑组织,采用酶消化、机械吹打、离心、培养原代神经干细胞克隆。②神经干细胞以2×108L-1密度接种培养,分4组,每组6瓶细胞,DMEM/F12组:加入DMEM/F12(1∶1)培养基含体积分数为0.1的小牛血清;碱性成纤维细胞生长因子组:培养基为含20μg/L碱性成纤维细胞生长因子;表皮生长因子组:培养基含30μg/L表皮生长因子;碱性成纤维细胞生长因子+表皮生长因子组:先用含碱性成纤维细胞生长因子的培养基培养2h后再换成含表皮生长因子的培养基培养。培养24h后更换培养瓶,培养14d后显微镜下计数原代克隆数,免疫组化法检测干细胞特殊标记蛋白巢蛋白的表达。主要观察指标:①神经干细胞的原代克隆。②单细胞克隆培养结果。③诱导分化结果。结果:①从胎鼠脑中分离的细胞具有连续传代形成克隆的能力,免疫荧光化学显示细胞球内细胞巢蛋白表达阳性。②采用含碱性成纤维细胞生长因子和表皮生长因子培养基先后作用培养的方法形成神经干细胞克隆率最高(0.630%)。③培养的神经干细胞克隆能诱导分化成神经元和神经胶质细胞。结论:①结果证实培养分离的细胞是神经干细胞。②采用含碱性成纤维细胞生长因子和表皮生长因子培养基先后作用培养的方法是获得神经干细胞的有效方法。
Abstract BACKGROUND: An effective method for obtaining neural stem cells in vitro is a very interesting issue in experimental stem cell research. Objective: To observe the effective method of culturing neural stem cells in vitro with medium containing different growth factors. Design: Single sample observation. Unit: Cell Biology Laboratory, School of Life Sciences, Sichuan University. MATERIALS: Ten pregnant 14dSD rats, DMEM / F121:1, basic fibroblast growth factor, epidermal growth factor, nestin antibody IgG, β-tubulinⅢ antibody IgG, glial fibrillary acidic protein antibody IgG, Secondary and secondary antibody labeled, fluorescein isothiocyanate labeled secondary antibody. Methods: The experiment was performed in the laboratory of Cell Biology, Sichuan University, 1999/2001. ① brain tissue removed from fetal rat forebrain, using enzyme digestion, mechanical blow, centrifugal, primary neural stem cell cloning. (2) Neural stem cells were inoculated at a density of 2 × 108L-1 and divided into 4 groups with 6 bottles of each group. DMEM / F12 group: DMEM / F12 (1: 1) medium supplemented with a volume fraction of 0.1; Fibroblast growth factor group: medium containing 20μg / L basic fibroblast growth factor; epidermal growth factor group: medium containing 30μg / L epidermal growth factor; basic fibroblast growth factor + epidermal growth factor group : First with basic fibroblast growth factor medium 2h and then replaced with epidermal growth factor-containing medium culture. The culture flasks were replaced after culturing for 24 hours, the number of primary colonies was counted under the microscope 14 days after culture, and the expression of special marker protein nestin in stem cells was detected by immunohistochemistry. MAIN OUTCOME MEASURES: ① primary cloning of neural stem cells. ② single cell cloning culture results. ③ induced differentiation results. Results: ① The cells isolated from the fetal rat brain had the ability of continuous passage to form clones, and immunofluorescence showed that the cell nestin expression was positive in the cells. ② The formation rate of NSCs was highest (0.630%) by using culture medium containing basic fibroblast growth factor and epidermal growth factor. ③ cultured neural stem cell clones can induce differentiation into neurons and glial cells. Conclusion: ①The results confirmed that the cultured cells were neural stem cells. ② using basic fibroblast growth factor and epidermal growth factor substrate culture method is to obtain an effective method of neural stem cells.