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建立大鼠胚胎神经干细胞克隆的实验

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14 d SD大鼠10只,DMEM/F12 11,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Ig G,胶质纤维酸性蛋白抗体Ig G,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(11)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2 h后再换成含表皮生长因子的培养基培养.培养24 h后更换培养瓶,培养14 d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.

Establishment of embryonic neural stem cell clone in rats following ordinal treatment of basic fibroblast growth factor and epidermal growth factor

BACKGROUND: The effective method to obtain neural stem cells through in vitro culture that deserves to pay more attention in experimental studies of stem cells.OBJECTIVE: To investigate the effective methods to culture neural stem cells in vitro using culture medium containing different growth factors.DESIGN: Single sample observation.SETTING: Laboratory of Cell Biology, College of Life Science, Sichuan University.MATERIALS: Ten SD rats which were pregnant for 14 days, DMEM/F12 1:1, basic fibroblasts growth factors, epidermal growth factor; nestin antibody IgG , β- microtubule protein Ⅲ antibody IgG , glial fibrillary acidic protein antibody IgG, biotin labeled second antibody and third antibody and fluorescinisothiocyate (FITC)-labeled second antibody were used in this experiment.METHODS: This experiment was carried out at the Laboratory of Cell Biology, College of Life Science, Sichuan University from 1999 to 2001. ① Brain tissue was taken out from the procerebrum of fetal rat, then primary neural stem cell clone was obtained through enzymatic digestion, mechanical treatment, centrifugation and culture. ② Neural stem cells were inoculated and cultured at 2×108 L-1 and divided into 4 groups with 6 bottles of cells in each group. DMEM/F12 (1:1 )culture medium containing 0.1 volume fraction of fetal bovine serum was added , serving as DMEM/F12 group; culture medium containing 20 μg/L basic fibroblast growth factor was added , serving as basic fibroblast growth factor group; culture medium containing 30 μg/L epidermal growth factor was added , serving as epidermal growth factor group; Culture medium containing basic fibroblast growth factors was added to culture for 2 hours, then culture medium containing epidermal growth factors was used for further culture, serving as basic fibroblast growth factor+ epidermal growth factor group. Culture flask was change after 24-hour culture; another 14 days later, primary clone number was counted under the microscope, and expressions of specially labeled albumen nidogen of stem cells were detected with immunohistochemistry.MAIN OUTCOME MEASURES: ① Primary clone of neural stem cells.② Clone culture result of unicell. ③ Induction and differentiation results.RESULTS: ① The cells isolated from the brain of fetal rats possess the ability to consecutively passage and form clone , and immunofluorescent staining showed cell nidogen expression positive in the cell sphere. ②Clone rate of neural stem cells was the highest (0.630%)using ordinal culture of basic fibroblast growth factor and epidermal growth factor. ③ The cultured neural stem cell clone can be induced and differentiated neurons and glinl cells.CONCLUSION: ① The results proved that the cultured and isolated cells are neural stem cells. ②Ordinal treatment of basic fibroblast growth factor and epidermal growth factor is an effective method to obtain neural stem cells.