5-氮杂胞苷通过p53／Gadd45α信号途径影响小鼠神经干细胞的增殖

The affect of 5-Azacytidine on proliferation of neural stem cells of mice via p53 /Gadd45α signaling pathway

目的:通过研究DNA甲基化抑制剂5-氮杂胞苷(5-Azacytidine,5-Aza)对小鼠神经干细胞(neural stem cells,NSCs)的影响,进一步阐明DNA甲基化模式的变化对神经发生的影响及机制。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5-Aza对NSCs增殖的影响;应用流式细胞仪检测5-Aza对NSCs细胞周期和凋亡的影响;并用RT-PCR、Western Blot法检测给予5-Aza对NSCs细胞p53和Gadd45αmRNA水平和蛋白表达的影响。结果:MTT法测定结果显示:与对照组相比,5-Aza能够明显抑制NSCs的增殖;流式细胞仪测定结果显示,5-Aza组NSCs在G0/G1期细胞数量较对照组明显减少(P<0.01),而S期、G2/M期细胞数量则明显增多(P<0.01),表明5-Aza可引起细胞周期在G2/M期停滞;且5-Aza组NSCs的凋亡率与对照组相比明显增加(P<0.01)。RT-PCR结果显示:5-Aza组NSCs中p53和Gadd45αmRNA水平较对照组明显增高(P<0.05);Western Blot结果也显示:与对照组相比,5-Aza组NSCs中p53和Gadd45α蛋白表达量明显增加(P<0.05)。结论:5-Aza可能通过影响NSCs细胞周期和诱导凋亡抑制小鼠NSCs增殖;5-Aza阻滞NSCs细胞周期的作用机制可能与p53/Gadd45α信号途径相关。

Objective: To explore the effects and mechanisms of 5-Azacytidine(5-Aza),a DNA methylation inhibitor on the proliferation of neural stem cells( NSCs) of mice in vitro,and to further elucidate effects and mechanisms of DNA methylation status changes on neurogenesis. Methods: Neurospheres were isolated from newborn mice brain and suspension cultured in serum-free medium. The effect of 5-Aza on proliferation of NSCs was detected by MTT. The effects of 5-Aza on apoptosis and cell cycle of NSCs were determined by flow cytometry. The mRNA levels of p53 and Gadd45α were assayed by RT-PCR. Western Blot were performed to investigate the expression of p53 and Gadd45α. Results: The MTT results showed that 5-Aza significantly inhibited NSCs proliferation compared with control group; the flow cytometry results revealed that the number of NSCs in G0 /G1 phase was obviously decreased,S and G2 /M phases were increased in 5-Aza-treated groups compared with control group(P < 0. 01),which indicated G2 /M phase attest after 5-Aza treatment; moreover,the apoptosis ratio of 5-Aza-treated NSCs was evidently higher than that of control group(P < 0. 01). RT-PCR results showed that the mRNA levels of p53 and Gadd45α in 5-Aza-treated NSCs were elevated compared with the control group(P < 0. 05); Western Blot results showed that the protein levels of p53 and Gadd45α in 5-Aza-treated NSCs were also increased compared with control group(P < 0. 05). Conclusion: 5-Aza inhibited the proliferation of NSCs via affecting cell cycle distribution and inducing NSCs apoptosis; the mechanism of 5-Aza caused cell cycle arrest may relate with p53 / Gadd45α pathway.