胎儿海马神经干细胞的体外培养及神经元前体细胞的纯化

目的　从人胎儿海马分离培养具有自我更新和多向分化能力的神经干细胞 ,并从中纯化神经元前体细胞。　方法　利用无血清培养从胚胎 4个月的胎儿海马区分离细胞 ,并在体外连续传代培养 ;采用免疫荧光细胞化学技术检测神经上皮干细胞蛋白 (nestin)的表达、经BrdU孵育后的BrdU表达 ;比较两种诱导分化方法所获得的神经元和胶质细胞的比例差异 ;利用单细胞克隆技术纯化神经元前体细胞。　结果　分离的细胞具有连续克隆能力 ,在体外培养了 16个月 ,传了 4 3代 ;细胞冻存、复苏后仍保持干细胞特性 ;培养的细胞呈Nestin阳性 ,在BrdU孵育后呈BrdU阳性 ,诱导分化后的细胞能够表达Tubulin、NeuN或GFAP ;利用无血清诱导所得到的分化细胞中神经元的比例约占 80 % ,而血清诱导的分化细胞中胶质细胞的比例则大于 90 %。利用单细胞克隆技术可从神经球中纯化的细胞表达Nestin ,并且全部分化成神经元。　结论　从胎儿海马区分离的细胞具有自我更新能力和多向分化潜能 ,属于神经干细胞 ,从中可纯化出神经元前体细胞

THE CULTURE OF NEURAL STEM CELLS FROM THE HIPPOCAMPUS IN HUMAN EMBRYO AND THE PURIFICATION OF NEURAL PRECURSORS

Objective To isolate and identify neural stem cells which have the ability of self|renewing and multipotential differentiation from the hippocampus of human embryo and to purify neuronal precusors from those cells. Procedures The neural cells were isolated from the hippocampus of human embryo by using serum|free culture.These cells were cultured and passaged continuously in vitro. The immunocytochemistry with fluorescence was used to detect antigen of nestin in the cultured cells and the immunocytochemistry of specific antigens(Tubulin,NeuN,GFAP) for mature neurons and glia was used for these cells after being induced and differentiated.The different effects of two methods of differentiation and induction were compared.The neuronal precursors from these cells were purified. Results The cells isolated from hippocampus of human embryo had been passaged in vitro at least 43 passages in 16 months.They expressed nestin and were also BrdU|positive after incubation in the medium with BrdU for 48 hours.They were shown to form clones and express tubulin,NeuN or GFAP after differentiation.The serum|free medium for differentiation and induction could induce 80% cells into the neurons and the medium with serum could induce 90% cells into the glial.The neuronal precursors could be purified by single cell|clone analysis.Conclusion The cells harvested from hippocampus in vitro were shown to have capabilities for self|renewing and multipotential differentiation and they should belong to the neural stem cells,from which neuronal precursors could be purified.