表皮生长因子和碱性成纤维细胞生长因子对人胎脑室下区神经干细胞增殖分化的影响

背景由于神经干细胞分化的多向性和不确定性给临床应用造成巨大困难,因此,探讨神经干细胞增殖分化条件已成为解决这一问题的关键.目的观察表皮生长因子和碱性成纤维细胞生长因子对人胎脑神经干细胞增殖分化的影响. 设计以培养的人胚神经干细胞为观察对象,随机对照实验.单位解放军第一军医大学珠江医院儿科.材料实验于2004-01/05在全军儿科中心实验室进行.随机在广州市某三级甲等医院产科选取自愿水囊引产的16周胎儿2例(胎儿父母签署同意书),取其脑室下区组织分别在无血清培养基和血清培养基进行细胞培养.方法采用含有表皮生长因子、碱性成纤维细胞生长因子以及表皮生长因子+碱性成纤维细胞生长因子的无血清培养基对人胎脑室下区组织进行原代细胞培养;两种生长因子的浓度均为20 μg/L,采用血清培养基对原代培养的细胞克隆球进行分化实验;并采用免疫组化技术对分化细胞进行检测.主要观察指标各组克隆球数量及其分化为神经元和星形胶质细胞的变化.结果①碱性成纤维细胞生长因子组、表皮生长因子+碱性成纤维细胞生长因子组形成的原代克隆球数量无显著差异(150.3±14.9),(173.6±26.4)个/孔,P＞0.05],但均明显多于表皮生长因子组(99.5±14.9)个/孔,P＜0.01].②碱性成纤维细胞生长因子组以及表皮生长因子+碱性成纤维细胞生长因子组克隆球分化为神经元的数量明显多于表皮生长因子组,而表皮生长因子组产生较多的星形胶质细胞.结论①碱性成纤维细胞生长因子能促进人胎脑室下区神经干细胞增殖,所形成的细胞克隆球能分化出较多的神经元.②两种因子合用并没有获得明显的优于单用碱性成纤维细胞生长因子的结果,提示不存在协同作用.

Effect of epidermal growth factor and basic fibroblast growth factor on neural stem cells isolated from subventricular zone of human embryonic brain

BACKGROUND: Pleiotropia and indeterminateness of the differentiation of neural stem cells (NSCs) cause great difficulties for clinical application.Therefore, it is the key to solving the problem to investigate the proliferation and differentiation condition of NSCs.OBJECTIVE: To observe the effects of epidermal growth factor (EGF)and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of neural stem cells of human embryonic brain.DESIGN: A randomised and controlled experiment taking cultured human embryonic stem cells as subjects.SETTING: Department of Pediatrics of Zhujiang Hospital, the First Military Medical University of Chinese PLA.MATERIALS: This experiment was conducted at the Central Laboratory of the Department of Pediatrics of Chinese PLA from January to May 2004.Two 16-week embryos from induced labor by water bags voluntarily were chosen at random from the Department of Obstetrics in a tertiary hospital in Guangzhou (Informed consent was obtained from the parents of the fetuses). Then, cells in the subventricular zone were cultured in serum-free medium and serum medium, respectively.METHODS: Primary cells of subventricular zone of human embryonic brain were cultured with serum-free nedium containing EGF, bFGF and EGF + bFGF. The concentration of two growth factors was both 20 μg/L;experiment of differentiation was performed on the cell neurospheres cultured from the primary generation with serum culture medium; differentia tive cells were detected with immonohistochemical technique.MAIN OUTCOME MEASURES: The number of neurospheres and the change of neurons and gliocytes from neurospheres in each group.RESULTS: ① There was no significant difference in the number of primary neurospheres between bFGF group and EGF+bFGF group (150.3±14.9) /well vs (173.6±26.4)/we11, P ＞ 0.05] , but the number in the two groups was both more than that in EGF group (99.5±14.9)/we11, P ＜ 0.01].② The neurons differentiated from neurospheres in bFGF group and EGF+bFGF group outnumbered those in EGF group, but gliocytes in EGF group outnumbered those in EGF + bFGF group.CONCLUSION: ① bFGF can promote the proliferation of neural stem cells of subventricular zone of human embryonic brain, and the formed neurospheres can differentiate more neurons. ② Combination of EGF and bFGF is not superior to single bFGF in effect, suggesting that there is no synergistic effect.