摘要
目的:通过检测5-氮杂胞苷作用于急性早幼粒细胞白血病细胞系(HL60)细胞前后P16、死亡相关蛋白激酶(Deathassociated protein kinase,DAPK)及6-氧-甲基鸟嘌呤-DNA甲基转移酶(O-6-methylguanine-DNA methyltransferase,MGMT)基因甲基化状态的改变及基因表达的变化情况,探讨5-氮杂胞苷对髓系白血病细胞系作用的相关机制。方法:采用常规细胞培养,加入5-氮杂胞苷培养48 h后,以甲基化特异性PCR(Methylation specific PCR,MSP)检测基因甲基化状态,以RT-PCR检测药物作用前后基因表达水平。结果:MSP显示用药前P16基因、DAPK基因及MGMT基因呈高甲基化状态,应用5-氮杂胞苷处理后P16及DAPK基因发生了完全去甲基化,MGMT基因在用药后发生了部分去甲基化;应用5-氮杂胞苷处理前后P16、DAPK及MGMT基因表达强度分别是(0.22±0.04)与(0.64±0.12)、(0.36±0.08)与(1.27±0.09)及(0.75±0.1)与(1.37±0.1),3个基因前后表达强度均有统计学差异(P<0.05)。结论:5-氮杂胞苷通过对P16、DAPK及MGMT甲基化基因的去甲基化作用,使这些基因恢复表达,从而抑制髓系白血病细胞系的增殖,发挥肿瘤细胞增殖抑制作用。
Objective：To explore the effect of 5-azacytidine on myeloid leukemia cell lines through detecting methylation state and expression changes of P16,death associated protein kinase （DAPK） and O-6-methylguanine-DNA methyltransferase（MGMT） after 5-azacytidine treatment for acute promyeloeytic leukemia cell lines（HL60 ceils）. Methods：HL60 cells were cultured with 5-azacyti- dine by routine method for 48 h. Then methylation-specific PCR（MSP） and RT-PCR were respectively used to detect gene methyla- tion status and expression changes of P16, DAPKand MGMT before and after being treated with 5-azacytidine. Results：MSP showed hypermethylated P16 ,DAPK and MGMT genes before 5-azacytidine treatment. Complete demethylation was observed in P16 and DAPK genes and partial demethylation was observed in MGMT gene after HL60 cells being co-cultured with 5-azacytidine. Expression intensities of P I6, DAPK and MGMT genes in HL60 ceils before and after treatment of 5-azacytidine were （0.22 ± 0.04） vs. （0.64 ± 0.12）, （0.36 ± 0.08） vs. （1.27 ± 0.09） and （0.75 ± 0.1 ） vs. （1.37 ± 0.1） respectively（P〈0.05）. Conclusions：The CpG islands of P16, DAPK and MGMT genes are hypermethylated in HL60 cells. 5-azacytidine inhibits proliferation of leukemic cells by demethylation of some related genes.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2012年第10期864-867,共4页
Journal of Chongqing Medical University
基金
重庆市卫生局科研基金资助项目(编号:渝卫科教34号06-2-193)
