摘要
目的探讨人胚胎干细胞分化为神经干细胞过程中,经拟胚体(embryonic body,EB)法和直接分化法的不同效率。方法人胚胎干细胞常规培养消化后,分为两组:A组,经EB法分化;B组,添加noggin和ITSFn直接分化法。倒置相差显微镜观察细胞形态变化,RT-PCR检测细胞各阶段标志物,免疫荧光及流式细胞仪观察两组细胞Nestin阳性细胞率。神经干细胞继续分化,免疫荧光、RT-PCR法检测MAP2、GFAP表达。结果RT-PCR检测到OCT4、nestin表达。B组nestin阳性细胞率明显高于A组,差异有统计学意义(P<0.01),且诱导周期短于A组。神经干细胞继续分化,得到不同数量的神经元和胶质细胞,MAP2、GFAP分别阳性。结论在体外采用定向分化诱导,人胚胎干细胞不经EB,可直接定向分化为神经干细胞,且诱导效率比EB法高。因此直接分化法是一种经济实用的诱导方法。
Objective To investigate the feasibility and efficiency of differentiation of human embryonic stem cells （hESCs） into neural cells with or without a period of embryonic body （EB） culture in vitro. Methods The hESCs were digested and divided into 2 groups： group A underwent EB culture, while cells in group B were induced by noggin and ITSFn directly without EB culture. Morphologic changes were observed under inverted microscope. The different markers were detected by RT-PCR. Nestin was detected by immunofluorescence and flow cytometry after hESCs differentiated. In the further differentiation of neural stem cens, MA/P2 and GFAP were observed by immunofluorescence and RT-PCR. Results In group B, the positive rate of nestin was higher than that in group A （P〈0. 01） . The cycle time of group B was shorter than that of group A. MAP2 and GFAP were detected after the differentiation of neural stem cells. Conclusion The hESCs can differentiate into neural cells without EB culture in vitro. This method is more efficient than the traditional.
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2009年第3期233-237,共5页
Chinese Journal of Histochemistry and Cytochemistry
关键词
人胚胎干细胞
细胞分化
神经干细胞
Human embryonic stem cell
Cell differentiation
Neural stem cell
