全反式维甲酸对HL-60细胞分化和凋亡的影响

Effect of All Trans Retinoid Acid (ATRA) on Differentiation and Apoptosis of HL-60 Cell

背景与目的:用全反式维甲酸(alltransretinoidacid,ATRA)治疗早幼粒细胞性白血病(premyeloidleukemia,M3)在肿瘤治疗史上是一个重要里程碑。已有研究表明,ATRA治疗白血病的机理是诱导白血病细胞向成熟细胞方向分化,然而人们对分化的肿瘤细胞最终去向还不完全清楚。本实验进一步探讨ATRA诱导HL-60细胞分化和凋亡的关系。方法:以分化诱导剂ATRA(终浓度为10μmol/L)作用不同时间的HL-60细胞为检测对象,应用流式细胞仪分析细胞表面的分化标志物及细胞周期;经碘化丙啶(propidiumiodide,PI)染色后,激光共聚焦显微镜下对已分化细胞进行形态学确认;应用流式细胞仪检测药物诱导后不同时间点的细胞凋亡。结果:(1)随着药物诱导时间的延长,被诱导的HL-60细胞体积逐渐增大,在72h后,被诱导细胞开始表达分化标志物CD11b并出现细胞核型的变化;(2)在药物诱导96h后,细胞开始出现了凋亡峰,而在药物诱导72h后脱药培养8h,则可以出现高于96h的凋亡峰。结论:ATRA并不能诱导HL-60细胞完成终端分化,但是该药可以诱导白血病细胞向成熟方向分化,而且已分化的肿瘤细胞于发生凋亡。

BACKGROUND &OBJECTIVE:Treatment of premyeloid leukemia with all trans retinoid acid (ATRA) is a milestone in the history of chemotherapy of malignant tumor. Previous studies suggested that the mechanism of treating premyeloid leukemia with ATRA is inducing premyeloid leukemia cells to differentiate along myelocyte lineage, but the fate of differentiated tumor cells is not clear. This study was designed to investigate the relationship between the differentiation of HL 60 induced by ATRA and apoptosis. METHODS:HL 60 cells influenced by ATRA (10 μmol/L) capable of inducing differentiation for different time were used as the subject. The differentiation marker on the cell surface and cell cycle were analyzed using flow cytometry. The differentiated cells were identified by confocal microscope after having been stained with propidium iodide (PI). Meanwhile,the changes of the apoptosis of the cells induced by ATRA at different time were analyzed using flow cytometry. RESULTS: (1)With drug inducing time increasing, the volume of the differentiated cells was enlarged gradually. After 72 hours, the differentiated cells began to express differentiating marker CD11b and the nuclei morphology of the differentiated cells was changed. (2)After 96 hours of drug inducing, the induced cells began to show apoptosis peak, but when the cells was washed once after 72 hours of drug inducing with RPMI 1640 medium and resuspended in RPMI 1640 medium supplemented with 10%fetal calf serum and then cultured in 5%CO2, 37℃for 8 hours,the cells began to show apoptosis peak,and the apoptosis peak was higher than that of the cells after 96 hours of drug inducing. CONCLUSION: ATRA cannot induce HL 60 to achieve terminal differentiation,but the differentiation of HL 60 can be induced by ATRA and the differentiated leukemia cells are easy to apoptosis.