STI571诱导K562细胞耐药机制的实验研究

Study on the resistant mechanisms of K562 cells induced by STI571

目的体外建立对STI571耐药的K562细胞系(简写为K562-R),并对其耐药机制进行初步分析。方法用药物浓度逐步递增的方法培养野生型K562细胞(简写为K562-W)。绘制K562-R生长曲线,MTT法检测STI571、阿霉素(adriamycin,ADR)和三尖杉酯碱(harringtonine,HT)对K562-R的细胞毒作用,Ho-echst33342染色检测STI571对K562-R诱导凋亡作用。流式细胞术检测K562-R的MDR-1表达情况;基因测序检测K562-R的Abl激酶ATP结合区点突变情况;间期荧光原位杂交(interphasal fluorescence in situ hybridization,I-FISH)检测Bcr/Abl融合基因扩增情况。结果生长曲线绘制结果表明K562-R在0.5μmol/L的STI571环境中可呈指数增长;MTT法检测STI571对K562-W和K562-R的半数抑制浓度(50% inhibiting concentration,IC50)分别为(1.64±0.13)μmol/L和(3.32±0.05)μmol/L,K562-R的耐药倍数为(2.04±0.16)倍,而ADR和HT对两种细胞毒性作用均呈剂量依赖性反应,耐药前后差异无显著性(P值分别为0.472和0.108);Hoechst33342细胞凋亡试验表明STI571对K562-W诱导凋亡作用呈剂量依赖性,对K562-R作用减弱。流式细胞术检测K562-R和K562-W的MDR-1表达率分别为2.68%和1.39%(P<0.001),基因测序表明两种细胞Abl激酶ATP结合区序列完全一致;I-FISH检测初步观察K562-R的Bcr/Abl融合信号拷贝数增多(P<0.001)。结论体外成功建立STI571耐药的K562细胞系,其耐药机制可能与Bcr/Abl融合基因扩增有关。

Objective To establish a cell line with resistance to STI571 （K562 - R） in vitro by culturing the wild -type K562 ceils（KS62 - W） and explore the potential mechanisms of acquired resistance. Methods K562 - W ceils were cultured in gradually increased concentrations of STI571. The growth curve was drawn by counting cells. The cytotoxic effects of K562 - W and K562 - R were analyzed by MTT assay and apoptosis was detected by Hoechest33342 staining. MDR -1 expression examining, sequence analysis and interphasal fluorescence in situ hybridization （I -FISH） were used to study the potential mechanisms of acquired resistance. Results K562 - R showed exponential growth in 0.5 μmol/L STI571. 50% inhibiting concentrations（ IC50 ） of K562 -R and K562 -W（ 1.64 ± 0. 13 ） μmol/L and （3.32 ± 0. 05 ） μmol/L respectively by MTr assay and there was （2.04 ± 0. 16） fold resistant to STI571. The comparison of the cytotoxic effects of K562 - W and K562 - R treated with different concentration of adriamycin（ADR） or harringtonine （HT） shows no statistic difference. The apoptosis percentage of K562 - R treated with different concentration of STI571 significantly decreased as compared with that of K562 - W. The MDR - 1 expression percentages of K562 - R and K562 - W with FASC analysis were 2.68% and 1.39% respectively（P 〈0. 001 ）. No point mutant in the Bcr/Abl ATP - binding site was detected and the copies of Bcr/Abl fusion gene were found to increase in K562 - R by I - FISH analysis（P 〈 0. 001 ）. Conclusion In vitro, we establish a resistant cell line. The resistant mechanism of K562 - R involves amplification of Bcr/Abl fusion gene.