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

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

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 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（P0.01）;the ratio of NSCs in G0/G1 phase was decreased（P0.01） and G2/M phase was increased in folic acid group compared with control group（P0.01）;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.