Objective: To investigate the effect of serum on the differentiation of neural stem cells.
Methods: The neural stem cells were isolated from the embryonic hippocampus tissues of Sprague Dawley rats at 14 day of pregnancy. After culturing and passaging, the 3rd generation cells were identified by immunocytochemical staining. Then, the cells were divided into 3 groups according to the concentrations of fetal bovine serum (FBS) used in the differentiation cell culture medium: 5% (group A), 1% (group B), 0 (group C), respectively. The other components of the culture media in 3 groups were the same. Cell viability was determined by using the Live/Dead cell staining at 8 days; the expressions of glial cell marker [glial fibrillary acidic protein (GFAP)] and neuronal marker (β-Ⅲ Tubulin) were determined and analyzed by immunocytochemical staining and real-time fluorescent PCR at 4 and 8 days of culture.
Results: Based on cell morphology and immunocytochemical staining, neural stem cells were identified. Cells were growing well with no death in all groups. With decreasing FBS concentration, the expression of GFAP was significantly decreased on both protein and mRNA level, whereas the expression of β-Ⅲ Tubulin was evidently increased. The staining of each group at 8 days was more obvious than that at 4 days. There were significant differences in mRNA expressions of GFAP and β-Ⅲ Tubulin at 4 and 8 days between groups ( P<0.05).
Conclusion: Serum can promote the differentiation of neural stem cells into glial cells. At the same time, it inhibits the differentiation of neural stem cells into neurons, the lower the serum concentration, the smaller the effect.
目的: 探讨血清对神经干细胞分化过程的影响。.
方法: 取孕 14 d SD 大鼠胚胎脑组织，分离培养神经干细胞并传代。取第 3 代细胞倒置相差显微镜下观察细胞形态，并行巢蛋白（Nestin）免疫细胞化学染色鉴定。将鉴定后的细胞分为 A、B、C 3 组，各组细胞培养基除 FBS 浓度不同（分别为 5%、1%、0）外，其余成分均一致。培养第 8 天，行活/死细胞染色观察细胞生长情况；培养第 4、8 天，行免疫细胞化学染色以及实时荧光定量 PCR 检测，观察胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）、Ⅲ 型 β 神经元微管蛋白（β-Ⅲ Tubulin）表达。.
结果: 经细胞形态及免疫细胞化学染色鉴定培养细胞为神经干细胞。培养第 8 天，活/死细胞染色示 A、B、C 组细胞均无死亡现象。免疫细胞化学染色示，培养第 4、8 天，A、B、C 组 GFAP 蛋白表达随 FBS 浓度降低而逐渐减弱，但 β-Ⅲ Tubulin 表达逐渐增强；各组第 8 天时染色均较第 4 天时增强。实时荧光定量 PCR 检测示，培养第 4 天和第 8 天 3 组间 GFAP mRNA及 β-Ⅲ Tubulin mRNA 相对表达量比较，差异均有统计学意义（ P<0.05）。.
结论: 血清能促进神经干细胞向胶质细胞分化，并减缓其向神经元分化速度，且血清浓度越低，该影响越小。.
Keywords: Neural stem cell; cell differentiation; glial cells; neurons; rat; serum.