人胎脑神经球及其构成细胞的成熟表型标志

目的 观察体外培养人胎脑神经干细胞球的形成，并鉴别球内是否有成熟的神经细胞存在，证实神经球的异质性。方法用添加表皮生长因子(EGF，20ng／mL)和成纤维细胞生长因子2(FGF2，10ng／mL)的无血清N2培养基，从人胎脑培养获得神经干细胞球，在倒置显微镜下观察其形成过程，用间接免疫荧光技术检测神经球的细胞表型标志神经丝（NF)、胶质纤维酸性蛋白(GFAP)、半乳糖脑苷脂(GalC)和巢蛋白(nestin)的表达，并计算出阳性细胞球的百分比。结果前6d，培养中形成大量的细胞集落，检测的细胞标志中，只有nestin ，其余标志均为阴性。培养至12d左右，细胞球的数量减少约1／3．球体的形态和大小也不相同。培养30d的神经球中，69．2％神经球为NF ，81．8％为GFAP ，100％为nestin ．未观察到GalC 。结论神经球有个成熟过程，神经球之间具有异质性,球内的细胞构成也具有异质性。

Neurospheres derived from human embryonic brain and mature cytophenotypic markers of neurosphere-forming cells

Objective To observe the formation of neurospheres ge nerated by culturing the neural stem cells from the human embryonic brain iden tify whether the neurosphere-forming cells express the mature neural cellular ma rkers, confirm the heterogeneity of neurospheres. Methods Human neurospher es generated by culturing neural stem cells from the human embryonic brain were obtained in the N2 media containing epidermal growth factor (EGF, 20 ng/mL) and fibroblast growth factor-2 (FGF2, 10 ng/mL), were observed in the formation prog ress by means of invert-microscopy, and the markers, including nerve filament (N F), glial fibrillary acidic protein (GFAP), galactocerebroside (GalC) and nestin , within the neurospheres were identified with indirect immuno-fluorescent techn iques. The percentages of the positive neurospheres were respectively calculated . Results A number of cell clusters expressing only nestin+,the three othe r markers negative, were formed in 6 d cultures. The cell sphere number decrease d by about 1/3 in 12 d, and the shape and the size of neurospheres changed signi ficantly. In 30 d cultures, the neurospheres demonstrated that all detected sphe res were nestin+ and GalC-, and 69.2% were NF+, 81.8% were GFAP+. Conclusion Neuroshpheres need a mature process. Both the neurospheres and the neurosphere -forming cells are heterogeneous.