不同时间点人胚胎脑神经干细胞分化为神经元和胶质细胞比率的研究

目的 比较不同时间点人胚胎脑神经干细胞分化为神经元和胶质细胞的比例.方法 取胎龄8～12 周的人胚胎脑海马,机械分离成单细胞悬液,以2×106 个细胞/ml接种到含h-EGF、h-bFGF和h-LIF的DMEM/F12培养液中.用1?S诱导神经干细胞球分化,12 h后行RNA-结合蛋白(musashil)免疫细胞化学鉴定,分别在2、5、7、14和23 d时行β-Ⅲ微管蛋白(β-Ⅲ Tubulin/Tuj1)、胶质原纤维酸性蛋白(GFAP)和半乳糖脑苷脂(galc)免疫细胞化学鉴定.结果 诱导分化12 h时细胞球中绝大部分细胞为Musashil阳性细胞,第2 d时有少量的β-ⅢTubulin和GFAP阳性细胞,未见galc阳性细胞;第7 d时β-Ⅲ Tubulin阳性细胞数目最多,约为细胞总数的48.2%,以后逐渐下降,而GFAP阳性细胞数目在第2～23 d内呈逐渐增加趋势,到第23 d时达细胞总数的65.3%;随分化时间延长,只能检测到极少量的galc阳性细胞.结论 1?S可促使人神经干细胞分化为中枢神经系统的3种主要细胞类型,且其分化为神经元和胶质细胞的比例在不同时间点上有显著差异.

RATIO OF NEURON AND GLIA DIFFERENTIATED FROM THE EMBRYONIC HUMAN NEURAL STEM CELLS AT DIFFERENT TIME IN VITRO

Objective To compare the ratios of neuron and glia differentiated from the embryonic human neural stem cells(hNSCs) at different times in vitro. Methods The cells from hippocampus of aborted human embryo tissue(8-12 weeks after conception) were mechanically dissociated and cultured with DMEM/F 12 medium containing h-EGF, h-bFGF, h-LIF. The NSCs were induced to differentiate by 1%FBS and identified by Musashil, β-Ⅲ Tubulin, GFAP and Galc immuno-cytochemical staining at different times. Results The cell spheres were Musashil positive at the 12th hour after differentiation.On the 2nd day after differentiation,a few cells exhibited β-Ⅲ Tubulin and GFAP immunoreactivities,but no Galc positive cells.The number of β-Ⅲ Tubulin immuno-positive cells reached the highest on the 7th day and the percent was 48.2%.The number of GFAP immuno-positive cells increased from the 2nd to the 23rd day and the positive ratio reached 65.3%.A few Galc immuno-positive cells were detected with time passing. Conclusion 1% FBS may promote human NSCs to differentiate into three cell types of central nervous