地西他滨对急性T淋巴细胞白血病细胞株Molt4细胞的作用及其机制研究  

Effects of decitabine against acute T lymphoblastic leukemia cell line Molt4

作　　者：刘进[1] 黄崇媚[1] 程辉[1] 唐古生[1] 胡晓霞[1] 周虹[1] 王健民[1] 杨建民[1] 

机构地区：[1]第二军医大学附属长海医院血液科,上海200433

出　　处：《中华血液学杂志》2015年第3期230-234,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金（81172249、81470322）

摘　　要：目的 探讨去甲基化药物地西他滨对急性T淋巴细胞白血病细胞株Molt4细胞的影响及其可能的作用机制.方法 CCK-8法检测地西他滨对细胞增殖的影响;Annexin Ⅴ/PI双重染色法检测地西他滨对细胞凋亡的影响;PI染色法检测细胞周期变化;转录组高通量测序筛选组间差异基因;亚硫酸氢盐测序法检测地西他滨作用Molt4细胞前后乳铁蛋白（LTF）基因启动子CpG岛的甲基化水平变化;实时定量RT-PCR及Western blot法分别检测LTF mRNA及蛋白的表达变化.结果 地西他滨能有效抑制Molt4细胞的增殖,抑制率呈时间和剂量依赖性增加,并能诱导细胞凋亡,使细胞阻滞于G0/G1期.0.50 μmol/L地西他滨处理72 h后,Molt4细胞LTF基因启动子甲基化率较对照组降低（45.0％对72.3％）,差异有统计学意义（P＜0.05）,mRNA和蛋白表达增加（P值均＜0.05）,同时还检测到caspase 3、caspase 9凋亡蛋白表达增加（P值均＜0.05）.结论 地西他滨诱导Molt4细胞凋亡,阻滞细胞周期在G0/G1期,抑制细胞增殖.LTF基因是地西他滨作用的重要靶点.Objective To explore the effects and possible mechanisms of decitabine on Molt4 in vitro.Methods Effects of decitabine on cells proliferation were detected by using CCK-8,the apoptosis by Annexin Ⅴ-FITC,cell cycles by propidium iodide-FACS.Discrepancy genes were screened by RNA-seq technique.The CpG methylation of lactoferrin （LTF） gene in Molt4 cells were identified by Bisulfite sequencing PCR （BSP）.The expression of LTF mRNA in Molt4 by RT-PCR and LTF protein expression were analyzed by Western blot.Results Decitabine effectively inhibited proliferation and induced apoptosis for Molt4 cells by an time-and dose-dependent manners.Cell cycles were arrested at the G0/G1 phase.The promoter methylation degree of LTF gene in Molt4 cells was 72.3% before decitabine treatment and decreased to 45.0% after treatment with 0.50 μmol/L decitabine for 72 h.After the reduction of methylation,expression of its mRNA and protein increased,meanwhile caspase 3 and caspase 9 protein expression levels increased.Conclusion The demethylating drug decitabine can induce apoptosis,detain cell cycle at phase G0/G1,inhibit proliferation and up-regulate LTF gene expression in Molt4 cells.LTF may become a new target for acute T lymphoblastic leukemia.

关 键 词：地西他滨 白血病 T细胞 Molt4细胞 

分 类 号：R733.71[医药卫生—肿瘤]