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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
OBJECTIVE: To isolate and culture neural stem cells capable of self-renewal and multidirectional differentiation from human fetal hippocampus and to purify neuronal precursor cells therefrom. Methods The cells were separated from the fetal hippocampus of fetal embryos at 4 months by serum-free culture and subcultured in vitro. The expression of nestin in neural epithelial cells was detected by immunofluorescence cytochemistry, and the expression of BrdU after BrdU incubation was compared. Differences in the ratio of neurons and glial cells between the two induced differentiation methods; Purification of neuronal precursor cells using single cell cloning techniques. RESULTS: The isolated cells had the ability of continuous cloning, cultured in vitro for 16 months and passaged for 4 3 passages. The cells remained frozen after resuscitation and maintained the characteristics of stem cells. The cultured cells showed Nestin-positive, BrdU-positive after BrdU incubation, and induced by BrdU Differentiated cells were able to express Tubulin, NeuN, or GFAP; the percentage of neurons in the differentiated cells was approximately 80% induced by serum-free induction, whereas the proportion of glial cells in serum-induced differentiated cells was greater than 90%. Nestin can be expressed from cells purified from neurospheres using single cell cloning techniques and all differentiate into neurons. Conclusion The cells isolated from fetal hippocampus have self-renewal ability and multi-directional differentiation potential, belonging to neural stem cells, from which neuronal precursor cells can be purified