体外诱导K562细胞伊马替尼耐药及其机理的初步探讨

Establishment of an Imatinib Resistance Cell Line K562R and Its Resistant Principia

目的体外诱导K 562细胞伊马替尼(im atin ib)耐药并对其耐药性进行检测。方法以浓度递增的方法诱导K 562对伊马替尼产生耐药,M TT法确定其耐药性。RT-PCR方法半定量测定耐药细胞中BCR/ABL基因水平,并进行BCR/ABL基因序列测定。流式细胞术测定P-gp表达。高效液相色谱仪(HPLC)测定耐药细胞内药物浓度。结果①浓度递增法成功诱导K 562细胞伊马替尼耐药(K 562R),K 562R细胞能长期在含5.0μm o l/L的伊马替尼的培养液中生长。②细胞生长曲线显示5.0μm o l/L伊马替尼作用于K 562R细胞120 h,其活细胞数量与0 h无明显差别。6个浓度梯度的伊马替尼(0、0.25、0.50、0.75、1.00、2.00μm o l/L)作用于K 562细胞24 h,发现所有浓度(除0μm o l/L外)伊马替尼均可抑制K 562细胞的生长,24 h细胞活力几乎为零。③M TT法抑制率测定K 562细胞半数抑制浓度约为0.75μm o l/L,K 562R细胞约为7.5μm o l/L,耐药倍数约为10倍。④HPLC细胞内药物浓度测定显示K 562R胞内伊马替尼药物浓度低于K 562细胞(P<0.01),流式细胞检测未发现P-gp表达。⑤374 bp的BCR/ABL基因序列分析未发现常规热点区域基因突变。结论体外成功诱导出伊马替尼耐药细胞——K 562R细胞。K 562R耐药主要环节为胞内药物浓度降低,而胞内药物浓度降低与P-gp无关。耐药机理需要进一步研究。

Objective To establish an imatinib resistance cell line and to study its resistant principia. Methods K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line. MTT assay, RT-PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance. Results （Dlmatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5 μmol/L. K562R cells were maintained in the media containing 5 μmol/L imatinib. ②Proliferation data showed that cell growth of K562R was not inhibited in 5 ⑷mol/ L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2.0 μmol/L imatinib. ③ The IC50 of K562R was about 7.5 μmol/L which was ten times higher than that of the parental cell. ④ HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells （up to 27.8-fold）. By flow cytometry, P-gp was not detected on K562R cell, indicating that low intracellular imatinib concentration may not be due to P-gp mediated efflux. ⑤ Sequence analysis of the 374 bp ABL kinase domain showed no mutation in K562R cell. Conclusion An imatinib resistance cell line K562R has been successfully established. Low intracellular imatinib concentration is a key link in the chain of cell resistance.