CAJ | 학술논문

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.
목적 탐토거갑기화약물지서타빈대급성T림파세포백혈병세포주Molt4세포적영향급기가능적작용궤제.방법 CCK-8법검측지서타빈대세포증식적영향;Annexin Ⅴ/PI쌍중염색법검측지서타빈대세포조망적영향;PI염색법검측세포주기변화;전록조고통량측서사선조간차이기인;아류산경염측서법검측지서타빈작용Molt4세포전후유철단백(LTF)기인계동자CpG도적갑기화수평변화;실시정량RT-PCR급Western blot법분별검측LTF mRNA급단백적표체변화.결과 지서타빈능유효억제Molt4세포적증식,억제솔정시간화제량의뢰성증가,병능유도세포조망,사세포조체우G0/G1기.0.50 μmol/L지서타빈처리72 h후,Molt4세포LTF기인계동자갑기화솔교대조조강저(45.0％대72.3％),차이유통계학의의(P＜0.05),mRNA화단백표체증가(P치균＜0.05),동시환검측도caspase 3、caspase 9조망단백표체증가(P치균＜0.05).결론 지서타빈유도Molt4세포조망,조체세포주기재G0/G1기,억제세포증식.LTF기인시지서타빈작용적중요파점.
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.