HL-60细胞内阿糖胞苷活性成分动态观察

摘要

目的 探索与研究建立白血病细胞内阿糖胞苷(Ara-C)活性成分及其代谢产物阿糖尿苷(Ara-U)水平测定方法,并观察白血病细胞内上述Ara-C 活性相关物质的变化规律与差异.方法 采用白血病细胞株(HL-60)及其耐药细胞株(HL-60/R),在Ara-C 作用下长期传代.对5、10、20、30、40、50 代细胞,分别采用放射免疫法(RIA)和高效液相色谱法(HPLC)动态观察和比较两种细胞内的Ara-C 活性成分,即三磷酸阿糖胞苷(Ara-CTP)、与DNA 嵌合的Ara-C 以及Ara-U的水平.结果 HL-60 细胞内Ara-CTP、与DNA 嵌合的Ara-C 在传至20 代后,均呈现持续下降;在细胞传代初期至第40代,各阶段HL-60 细胞内Ara-CTP 水平均显著高于HL-60/R;在传代初期至第20 代过程中,HL-60 和HL-60/R 细胞内的与DNA 嵌合的Ara-C 并无显著差异.经长期传代之后,HL-60 细胞内的Ara-U 呈持续上升,且均显著高于相同传代阶段的HL-60/R.结论 白血病细胞在Ara-C 的长期作用下,Ara-CTP、与DNA 嵌合的Ara-C 水平,在不同时段出现明显下降趋势,可能是导致白血病患者发生对Ara-C 耐药的主要原因.%Objectives To establish the methods for determination of the levels of cytosine arabinoside metabolites (Ara-CTP, DNA-incorporated Ara-C and Ara-U), and also observe the regularity and difference in change of these Ara-C metabolites in leukemia cells. Methods Radioimmunoassay and HPLC were used to determine the levels of Ara-C metabolites (Ara-CTP, DNA-incorporated Ara-C and Ara-U) in human promyelocytic leukemia HL-60 cells and its variant HL-60/ R cells. The changes of these metabolites in such two cell lines were also monitored and compared at different passages (5th, 10th, 20th, 30th, 40th and 50th passage). Results It showed that the concentrations of active metabolites of Ara-C (Ara-CTP and DNA-incorporated Ara-C) in HL-60 cells was constantly decreased after the 20 passages. The concentration of intracellular Ara-CTP in HL-60 cells was significantly higher than that in HL-60/R cells from 5th- to 40th- passage. It was found that there were no statistically significant differences in the concentration of intracellular DNA-incorporated Ara-C between HL-60 cells and HL-60/R cells from 5th- to 20th- passage. After long-term passage and culture, the Ara-U levels in HL-60 cells showed significant rising trend, and were significantly higher than that of the HL-60/R at different passages. Conclusions After long-term passage and culture, the intracellular active metabolites of Ara-C in leukemia cells, such as Ara-CTP and DNA-incorporated Ara-C, are significantly decreased at the different passages. This might be the main cause of Ara-C resistance in leukemia patients.