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目的探讨体外建立培养成体神经干细胞的方法。方法从6周龄大鼠脑组织中分离神经干细胞,用神经干细胞培养液(DMEM/F12培养液添加1%N2、2%B27、20μg/LEGF和20μg/LbFGF)使其稳定增殖,10%胎牛血清诱导其贴壁分化。倒置显微镜下观察神经干细胞形态学变化;流式细胞仪检测神经干细胞表面标记物巢蛋白(nestin),神经元特异性烯醇酶(neuron-specific enolase,NSE成熟神经元的特异性标志),半乳糖脑苷脂(galactocerebroside,Galc-C成熟少突胶质细胞的标记物)的表达。结果分离所得细胞能在体外传代培养,流式细胞仪检测发现Nestin阳性细胞为97.6%,细胞经胎牛血清诱导分化后能形成NSE、Galc-C阳性细胞。结论采用无血清的神经干细胞培养液能培养出具有分化潜能的成体神经干细胞。
Objective To investigate the method of culturing adult neural stem cells in vitro. Methods Neural stem cells (NSCs) were isolated from brain tissue of 6-week-old rats and stably proliferated in neural stem cell culture medium (1% N2, 2% B27, 20μg / L EGF and 20μg / L bFGF in DMEM / F12 medium) Bovine serum induces its adherent differentiation. Morphological changes of neural stem cells were observed under inverted microscope. Flow cytometry was used to detect the expression of nestin, neuron-specific enolase (specific marker of NSE mature neurons) on neural stem cells, Expression of galactocerebroside, a marker of Galc-C mature oligodendrocytes. Results The isolated cells could be subcultured in vitro. The number of Nestin positive cells detected by flow cytometry was 97.6%. NSE and Galc-C positive cells could be formed after the cells were differentiated by fetal bovine serum. Conclusion Adult neural stem cells with differentiation potential can be cultured using serum-free neural stem cell culture medium.