摘要
目的:探讨去甲基化药物地西他滨(Decitabine,DAC)的不同剂量对MDS-RAEB(骨髓增生异常综合征-原始细胞增多型)细胞株MDS-L细胞增殖的抑制作用及其相关作用机制。方法:采用LC-MS/MS生物分析法检测应用DAC 20和15 mg/m^2×5 d的两组MDS患者血药浓度水平;模拟患者的血药浓度,设计不同剂量的DAC(0.5、0.3、0.2、0.1、0.05、0.025和0.01μg/ml)作用于MDS-L细胞24、48、72和96 h,用CCK-8法检测细胞的增殖抑制效应;光学显微镜下观察细胞形态学变化;流式细胞术检测细胞周期变化及凋亡情况;PCR检测DAC作用后MDS-L细胞P15的甲基化水平的变化。结果:注射结束即刻,DAC 20 mg/m^2×5 d组患者血药浓度为174.08±80.15(84.7-311)ng/ml,明显高于15 mg/m^2×5 d组的89.87±32.94(43.2-165)ng/ml(P=0.014)。DAC对MDS-L细胞有明显增殖抑制作用,且呈时间浓度依赖关系(r=0.786),但DAC≥0.1μg/ml浓度时,各浓度对细胞的抑制作用无明显差异。DAC处理后G_1期细胞明显增多,而S期细胞明显减少。与对照组相比,MDS-L细胞经DAC 0.01、0.025、0.05、0.1、0.2μg/ml作用96 h后,P15INK4B表达均下降,但各浓度之间无显著差异(P>0.05)。结论:低浓度DAC对MDS-L细胞有明显增殖抑制作用,在0.1和0.2μg/ml浓度时,对MDS-L细胞的抑制作用达理想范围。DAC可以将MDS-L细胞阻滞在G_1期,阻止G_1期细胞向S期细胞转化。
Objective： To investigate the inhibitory effect of decitabine（ DAC） in various dosages on the proliferention of MDS-RAEB cell line MDS-L and its mechanism. Methods： LC-MS/MS method was used to test the blood DAC concentration of 2 groups of MDS patients being treated with DAC 20 and 15 mg/m^2 × 5 d. In according to the various blood DAC concentration levels,the MDS-L cells were treated with different DAC dosages for 24,48,72 and 96 h,respectively. The CCK-8 method was applied to determine the cell proliferation,the flow cytometry was used to analyze the cell cycle and cell apoptosis changes,the P15 INK4 B DNA methylation status was measured by methylation specific PCR using EZ DNA Methylation-Gold Kit. Results： The blood DAC concentration of MDS patients treated with DAC 20 mg/m^2 × 5 d was 174. 08 ± 80. 15（84. 7-311） ng/ml,which was significantly higher than 89. 87 ± 32. 94（43. 2-165）ng/ml for the group treated with 15 mg/m^2 × 5 d（P = 0. 014）. DAC could notably inhibit the proliferation of MDS-L cells,and the effect was in dose-and-time-dependent manner（ r = 0. 786）. However,when DAC concentration was ≥0. 1 μg/ml,the proliferation inhibition rates were not significantly different between various dosages. After DAC treatment,MDS-L cells in G1 phase increased notably,while cells in S phase decreased significantly. Also,the P15^（INK4B） DNA methylation status of MDS-L cells decreased after being treated with DAC for 96 h,but the difference was not significant between various dosages. Conclusion： DAC can significantly suppress MDS-L cell proliferation,block MDS-L cells in G1 phase and induce the apoptosis at low concentration（. 1. 2 μg/ml）.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2017年第5期1471-1476,共6页
Journal of Experimental Hematology
