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目的:研究叶酸对体外培养小鼠神经干细胞(neural stem cells,NSCs)增殖和分化的影响及作用机制。方法:采用无血清悬浮培养方法分离培养新生小鼠脑NSCs,通过MTT法检测叶酸对NSCs增殖的影响;撤除生长因子后,用含10%胎牛血清的培养基诱导分化培养6 d后,采用Tuj1(神经元标记物)和GFAP(胶质细胞标记物)免疫荧光双标记法检测叶酸对NSCs分化的影响;并应用流式细胞术、RT-PCR法检测给予叶酸对NSCs细胞周期、p53和p21(waf1/cip1)mRNA水平的影响。结果:与对照组相比,MTT法测定结果显示,叶酸组NSCs增殖能力明显增强;分化后免疫荧光双标法测定显示,叶酸组Tuj1阳性细胞的比率明显增加,且差异具有显著性(P<0.01);流式细胞仪测定结果显示,叶酸组NSCs在G0/G1期细胞数量明显减少(P<0.01),而G2/M期细胞数量明显增多(P<0.01);RT-PCR结果显示,叶酸组NSCs中p53和p21 mRNA表达量明显降低。结论:叶酸能促进NSCs增殖及向神经元分化;叶酸对NSCs增殖和分化的影响与调节NSCs细胞周期及p53/p21(waf1/cip1)信号转导途径相关。
Objective: To study the effect and mechanism of folic acid on the proliferation and differentiation of neural stem cells (NSCs) cultured in vitro. Methods: The NSCs of neonatal mice were isolated and cultured by serum-free suspension culture. The effect of folic acid on the proliferation of NSCs was detected by MTT assay. After the growth factors were removed, the cells were induced to differentiate with 10% fetal bovine serum for 6 days. The effect of folic acid on the differentiation of NSCs was detected by immunofluorescence double labeling with Tuj1 (neuron marker) and GFAP (glial marker). Flow cytometry and RT-PCR were used to detect the effect of folic acid on the cell cycle, p53 and p21 (waf1 / cip1) mRNA levels. Results: Compared with the control group, the results of MTT assay showed that the proliferative ability of NSCs in folic acid group was significantly enhanced. The ratio of Tuj1 positive cells in folic acid group was significantly increased by immunofluorescence double-labeled assay after differentiation (P < 0.01). The results of flow cytometry showed that the number of NSCs in folic acid group was significantly decreased in G0 / G1 phase (P <0.01), while the number of cells in G2 / M phase was significantly increased (P <0.01) The expression of p53 and p21 mRNA in folic acid group was significantly decreased. CONCLUSION: Folic acid can promote the proliferation of NSCs and differentiate into neurons. The effect of folic acid on the proliferation and differentiation of NSCs is related to the cell cycle of NSCs and the signal transduction pathway of p53 / p21 (waf1 / cip1).