体外诱导K562细胞尼洛替尼耐药及其机制的初步探讨  

K562 cell line resistance to nilotinib induced in vitro and preliminary investigation of its mechanism

作　　者：王季石[1] 杨畅[2] 方琴[3] 韦四喜[1] 陈埕[1] 杨远[1] 王娅婷[1] 胡秀英[1] 马丹[3] 

机构地区：[1]贵阳医学院附属医院血液科、贵州省暨贵阳医学院附属医院造血干细胞移植中心,550004 [2]贵州省暨贵阳医学院附属医院药学院 [3]贵州省暨贵阳医学院附属医院附属医院药剂科

出　　处：《中华血液学杂志》2012年第11期906-910,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金（81070444）;贵州省科技厅国际合作项目（黔科合外G[2012]7043）;贵州省社会攻关项目（黔科合SY字[2011]3012）;贵州省科技厅科技基金（黔科平台[2009]4007;黔科合J字[2009]2164）;贵州省省长基金（黔省专合字[2010]84）;贵阳市科技局科技基金（筑科合同[2012103]34）

摘　　要：目的体外建立对尼洛替尼（nilotinib）耐药的白血病细胞株，并初步探讨其耐药机制。方法以尼洛替尼浓度递增的方法诱导人白血病细胞系K562细胞对其产生耐药株，命名为K562-RN，MTr法确定其耐药倍数。流式细胞术检测细胞凋亡。采用荧光原位杂交（FISH）法检测K562-RN细胞中bcr-abl融合基因表达。实时荧光定量PCR（RQ—PCR）和Westernblot法检测bcr-abl、HO-1、mdrl、Bcl-2和caspase-3mRNA和相关蛋白在耐药和敏感细胞中的表达。结果成功建立了针对尼洛替尼耐药的人白血病细胞株K562-RN。尼洛替尼对K562、K562-RN细胞的半数抑制浓度（IC50值）分别为（12．320±1．720）μmol／L和（24．742±2．310）μmol／L，K562-RN细胞相对于K562细胞的耐药倍数为2．01倍，且对阿霉素、高三尖杉酯碱、鬼臼乙叉甙和伊马替尼具有不同程度的交叉耐药性。在不同浓度尼洛替尼诱导下，K562-RN细胞凋亡率均较K562细胞明显下降。FISH法分析结果显示K562-RN细胞中bcr—abl融合基因阳性率为92％。K562一RN细胞中bcr—ab1、HO-1mRNA及其相应蛋白高表达，而促凋亡基因caspase-3mRNA及蛋白呈低表达，与K562细胞相比表达水平差异有统计学意义（P〈0．05），多药耐药基因mdrl及其编码蛋白P—gP在K562-RN细胞中呈高表达。结论通过药物浓度递增法成功建立了对尼洛替尼耐药的人白血病细胞株K552一RN，初步探讨了其耐药机制可能与bcr-abl、HO-1及mdrl高表达，同时下调caspase-3mRNA及其蛋白的表达有关。Objective To establish a bcr-abl＋ cell line resistance to nilotinib, and to investigate the possible mechanisms of resistance. Methods K562 ceils were treated with gradually increasing concentrations of nilotinib to generate resistance cell line K562-RN. The folder of drug-resistance was evaluated by MTT assay. Cells apoptosis rate was detected by flow cytometry, the mRNA level of bcr-abl fusion gene by FISH, and the expression of apoptosis relative gene mRNA and protein （ such as bcr-abl, HO-1, mdrl, Bcl- 2 and caspase-3） by RQ-PCR and western blot. Results The resistant cell line K562-RN was successfully established, with 2.01 fold resistant to nilotinib compared with K562 cell line [ the IC50 value of nilotinib to K562 and K562-RN were （12.320±1. 720）μmol/L and （24.742 ±2. 310）μmol/L, respectively]. It also had the cross resistance to adriamycin, homoharringtonine, etoposide and imatinib. Treated with different concentrations of nilotinib, cell apoptosis rate of K562-RN was significantly lower than that of K562 cells. The rate of bcr-abl gene positive cells was 92% in K562-RN by FISH assay. The mRNA and protein levels of bcr- abl, HO-1 and mdrl expression up-regulated in K562-RN cells, while those of caspase-3 expression down- regulated, being significantly statistical difference when compared with K562 ceils （P 〈 0.05 ）. Conclusion Human leukemic cell line resistance to nilotinib, K562-RN is established successfully by gradually increasing concentrations of drug. The mechanisms of resistance in K562-RN is probably associated with increasing expression of bcr-abl , HO-1, mdrl and decreasing expression of caspase-3 mRNA and protein levels.

关 键 词：尼洛替尼 K562细胞 抗药性 基因 HO-1 

分 类 号：R733.7[医药卫生—肿瘤]