CAJ | 학술논문

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,MTT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Western blot法检测bcr-abl、HO-1、mdr1、Bcl-2和caspase-3 mRNA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-RN细胞中bcr-abl、HO-1 mRNA及其相应蛋白高表达,而促凋亡基因caspase-3 mRNA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mdr1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K562-RN,初步探讨了其耐药机制可能与bcr-abl、HO-1及mdr1高表达,同时下调caspase-3 mRNA及其蛋白的表达有关.
목적 체외건립대니락체니(nilotinib)내약적백혈병세포주,병초보탐토기내약궤제.방법 이니락체니농도체증적방법유도인백혈병세포계K562세포대기산생내약주,명명위K562-RN,MTT법학정기내약배수.류식세포술검측세포조망.채용형광원위잡교(FISH)법검측K562-RN세포중bcr-abl융합기인표체.실시형광정량PCR (RQ-PCR)화Western blot법검측bcr-abl、HO-1、mdr1、Bcl-2화caspase-3 mRNA화상관단백재내약화민감세포중적표체.결과 성공건립료침대니락체니내약적인백혈병세포주K562-RN.니락체니대K562、K562-RN세포적반수억제농도(IC50치)분별위(12.320±1.720) μmol/L화(24.742 ±2.310)μmol/L,K562-RN세포상대우K562세포적내약배수위2.01배,차대아매소、고삼첨삼지감、귀구을차대화이마체니구유불동정도적교차내약성.재불동농도니락체니유도하,K562-RN세포조망솔균교K562세포명현하강.FISH법분석결과현시K562-RN세포중bcr-abl융합기인양성솔위92％.K562-RN세포중bcr-abl、HO-1 mRNA급기상응단백고표체,이촉조망기인caspase-3 mRNA급단백정저표체,여K562세포상비표체수평차이유통계학의의(P＜0.05),다약내약기인mdr1급기편마단백P-gp재K562-RN세포중정고표체.결론 통과약물농도체증법성공건립료대니락체니내약적인백혈병세포주K562-RN,초보탐토료기내약궤제가능여bcr-abl、HO-1급mdr1고표체,동시하조caspase-3 mRNA급기단백적표체유관.
Objective To establish a bcr-abl + cell line resistance to nilotinib,and to investigate the possible mechanisms of resistance.Methods K562 cells 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,mdr1,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 bcrabl,HO-1 and mdrl expression up-regulated in K562-RN cells,while those of caspase-3 expression downregulated,being significantly statistical difference when compared with K562 cells(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,mdr1 and decreasing expression of caspase-3 mRNA and protein levels.