胎儿脑组织体外保存获取神经干细胞的时限

THE TIME LIMITATION OF in vitro INCUBATION FOR ISOLATING NEURAL STEM CELLS FROM HUMAN FETAL BRAIN

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 。

Objective To study the possibility of isolating neural stem cells from human fetal hippocampus in different time of in vitro incubation. Procedures The neural cells were isolated from the human fetal hippocampus and incubated in 4℃ Hank solution for 0h,4h,24h,48h,72h,96h respectively by using serum\|free medium.Then they were cultured and passaged continuously in vitro. The fluorescent immunocytochemistry was used for detecting antigen of nestin in the cultured multipotent cells and the specific antigens(Tubulin,GFAP) for mature neurons and glia after being induced to differentiate.The proliferative capacity of the cultured cells was analyzed by the uptake of BrdU.The characteristics between the cells in different groups were compared. Results The cells from fetal hippocampus could be cultured successfully and formed cellular spheres before 72h of postmortem.These cultured cells expressed nestin and were also BrdU\|positive after incubation in the medium with BrdU for 48 hours.The ratio between neurons and glial cells was no difference in each group after the differentiation\|induction.The number of cellular spheres in culture had a tendency of decreasing with the extension of incubation in Hank solution.Conclusion\ The neural stem cells could be isolated from the human fetal hippocampus when the brain tissues were incubated in 4℃ Hank solution for less than 72h of postmortem,but there was an inverse relationship between the number of incubated spheres with the time course of postmortem.