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目的 探讨人胎儿脑海马组织体外保存不同时限后培养获取神经干细胞的可行性。 方法 利用无血清培养 ,从死亡后孵育在 4℃Hank液 0h、4h、2 4h、4 8h、72h及 96h的胎儿海马分离细胞 ,在体外连续传代培养 ;采用免疫荧光细胞化学方法检测培养细胞的神经上皮干细胞蛋白 (nestin)表达、经BrdU孵育后的BrdU表达以及诱导分化后的神经元标记 (tubulin)或星型胶质细胞抗原 (GFAP)表达 ;比较不同时限的细胞在Nestin的表达、增殖能力和诱导分化后神经元及胶质细胞比例的差异。 结果 从保存在 4℃Hank液 72h以内的各组胎儿海马中均可获得具有连续增殖能力的神经球 ,培养出的细胞Nestin表达为阳性 ,通过BrdU的摄取表明细胞处于活跃的有丝分裂状态 ,在此期限内各组之间无明显差异 ;诱导分化后的神经元和胶质细胞比例在各组之间亦相似 ;但随着在Hank液中孵育时间的延长 ,所获得的神经球数量呈减少趋势 ;在 96h组则未能获得神经球。 结论 人胎儿海马组织在 4℃Hank液中孵育 72h内可培养出呈神经球样生长的神经干细胞 ,但神经球数量与孵育时间呈负相关
Objective To investigate the feasibility of culturing and obtaining neural stem cells in human fetus hippocampus after being stored at different time points in vitro. Methods Serum-free culture was used to separate cells from fetal hippocampus incubated at 4 ℃ for 4h, 4h, 24h, 48h, 72h and 96h in Hank’s solution at 4 ℃, and then subcultured in vitro. Immunofluorescence cytochemistry Nestin expression, BrdU expression after BrdU incubation, and induction of differentiated neuronal tubulin or astrocyte antigen (GFAP) expression were compared. Nestin expression and proliferation were compared between different time points Differences in ability and proportion of differentiated neurons and glial cells. RESULTS: Neurospheres with continuous proliferation were obtained from each group of fetal hippocampus stored in Hank’s solution at 4 ° C for 72 hours. Nestin was expressed as positive in cultured cells, indicating that the cells were in an active mitotic state by the uptake of BrdU There was no significant difference between the groups within the time limit; the proportion of neurons and glial cells after differentiation was also similar among the groups; however, the number of neurospheres obtained tended to decrease with the extension of incubation time in Hank’s solution In 96h group, neurospheres were not obtained. Conclusion Human fetal hippocampal neurons cultured in Hank’s solution at 4 ℃ for 72 h could induce neuroblastoma cells to grow neural stem cells, but the number of neurospheres was negatively correlated with the incubation time