三氧化二砷对白血病细胞EVI-1基因作用的研究

Effect of arsenic trioxide(As_2O_3) on EVI-1 gene in leukemia cells

目的:探讨三氧化二砷(arsenic trioxide,As2O3)对白血病细胞中EVI-1基因的作用。方法:采用1μmol/L的As2O3处理1例高表达EVI-1基因的急性单核细胞白血病患者的原代细胞,并用反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测细胞的EVI-1 mRNA表达率;同时采用RT-PCR检测人白血病细胞株K562、HL-60、U937和THP-1中EVI-1 mRNA基因的相对表达量。选取EVI-1 mRNA表达量最高的THP-1细胞株作为研究对象,经多位点PCR测序确认其包含EVI-1基因全长片段后,分别以1、3、5μmol/L的As2O3处理THP-1细胞株,观察细胞形态变化,CCK8检测细胞活力,流式细胞仪检测细胞凋亡率,RT-PCR检测EVI-1 mRNA表达率,蛋白质印迹法检测细胞内EVI-1蛋白的表达情况。结果:As2O3能下调原代细胞中EVI-1 mRNA,抑制THP-1细胞株增殖、诱导其凋亡,且呈剂量、时间依赖性;As2O3能下调THP-1细胞株EVI-1 mRNA、EVI-1蛋白的表达。结论:As2O3通过抑制白血病细胞EVI-1 mRNA、EVI-1蛋白的表达,从而促进其凋亡。

Objective： To investigate the effect of arsenic trioxide on EVI-1 gene in leukemia cells. Methods： Primary leukemia cells from an acute monocytic leukemia patient with high expression of EVI-1 geue were treated with 1 p.mol/L As203, and expression of EVI-1 mRNA was detected by RT-PCR . RT-PCR was used to detect the relative expression of EVI-1 mRNA in four leukemia cell lines： K562, HL-60,U937 and THP-1. THP-1 cell line with the highest expression of EVI-1 mRNA was selected as the research object. By multiple-site PCR sequencing, the full length fragment containing EVI-1 mRNA were confirmed, and THP-1 cells were treated with different concentrations of As203 （1,3, and 5 p, mol/L）. Changes of cell morphology was observed, and CCK8 was used to test cell viability, flow cytometry was used to assess apoptosis. EVI-1 mRNA expression was detected by RT-PCR and EVI-1 protein expression was determined by Western blot. Results： As203 down-regulated EVI-1 mRNA in primary leukemia cells, and inhibited cell proliferation, induced apoptosis in a time- and concentration-dependent manner, down-regulated EVI-1 mRNA and EVI-1 protein in THP-1 cell line. Conclusions： Arsenic trioxide induces leukemia cells apoptosis via inhibiting expression of EVI-1 mRNA and EVI-1 protein.