叶酸通过调节p53/p21(waf1/cip1)信号途径促进小鼠神经干细胞增殖和分化

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。

Folic acid promote proliferation and differentiation of neural stem cells via regulation p53/p21(waf1/cip1) pathway in mice

Objective:To explore the effects and mechanisms of folic acid on proliferation and differentiation of neural stem cells(NSCs) in vitro in mice.Methods: NSCs were isolated from newborn mice brain and suspension cultured in serum-free medium.The effect of folic acid on proliferation of NSCs was determinated by MTT method;the differentiation of NSCs was detected by immunofluorescence double labeled with Tuj1(marker of neuron) and GFAP(marker of glia) after 6 day culture in 10% fetal calf serum medium condition without growth factors.The cell cycle of NSCs was detected by flow cytometry and the mRNA levels of p53 and p21 were determinated by RT-PCR.Results: The proliferation rate of NSCs and Tuj1 positive cells were significantly higher in folic acid group than control group(P<0.01);the ratio of NSCs in G0/G1 phase was decreased(P<0.01) and G2/M phase was increased in folic acid group compared with control group(P<0.01);and the mRNA levels of p53 and p21 in NSCs were downregulated after folic acid treatment.Conclusion: Folic acid can promote the NSCs to proliferate and differentiate into neurons;the mechanisms of folic acid on proliferation and differentiation of NSCs are related with cell cycle and p53/p21(waf1/cip1) pathway.