人胚不同脑区神经前体细胞的分离培养及分化特性的比较

COMPARATIVE STUDY IN VITRO ON THE CULTURE,PROLIFERATION AND DIFFERENTIATION CHARACTERISTICS OF HUMAN NEURAL PROGENITOR CELLS DERIVED FROM DIFFERENT EMBRYONIC BRAIN REGIONS

目的研究人胚不同脑区神经前体细胞(neural progenitor cells,NPCs)培养及增殖分化特性。方法取14-17周人胚脑区组织,分为新皮质、纹状体、间脑、中脑、后脑和延髓组,悬浮培养。鉴定细胞球巢蛋白抗原的表达,分化及自我更新能力。观察各脑区培养细胞的生长、增殖状况。新皮质、纹状体及间脑来源的神经球分化后,运用免疫荧光细胞化学法比较神经元及星形胶质细胞的比例。结果各脑区培养出的悬浮细胞球巢蛋白抗原阳性,可分化为MAP2或GFAP阳性细胞,且BrdU掺入实验阳性。体外培养第3d,纹状体及间脑组均可见大量神经球,且纹状体组明显多于间脑组;新皮质组传代后可见较多神经球;其它组仅见个别神经球。新皮质、纹状体、间脑来源的NPCs诱导分化后,MAP2或GFAP阳性细胞率各组间比较差异无显著性。结论人胚不同脑区均可培养出NPCs,从易到难依次为纹状体、间脑、新皮质及其它脑区。新皮质、纹状体、间脑来源的NPCs体外分化比例一致。

Objective To investigate the culture, proliferation and differentiation of human neural progenitor cells derived from different embryonic brain regions. Methods The free-floating cells of neocortex, striatum, diencephalon, mesencephalon, metencephalon and myelencephalon from 14-17-week embryonic brains were cultured. The expression of nestin, self-renewal and multipotential property of the cell clusters were examinated. The growth and proliferation of the cells isolated from different brain regions were analysed. After the neurospheres from neocortex, striatum and diencephalon were induced, the percentage of neurons or astrocytes was investigated by immunocytochemical staining. Results Nestin and Brdu immunochemical staining was positive in the cell aggregations derived from different brain regions, and all of them could differentiate into MAP2 or GFAP positive cells. On day 3 of culture, many neurospheres were formed in diencephalon and striatum groups, but more in striatum. By contrast, much less neurospheres were formed in neocortex, and only few in other groups. After differentiation of neural progenitor cells from neocortex, striatum and diencephalon, the percentage of MAP2 or GFAP positive cells did not show significant difference among the groups. Conclusion In vitro, neural progenitor cells can be isolated from different embryonic brain regions. The rate of neurosphere formation is higher in striatum than in diencephalon, neocortex and other regions. Under the same culture conditions, the percentage of differentiated cells does not show difference in the neural progenitor cells from striatum, diencephalon and neocortex.