抑制NAMPT表达增强K562细胞对伊马替尼的敏感性及相关机制研究  

Suppression of NAMPT Expression Enhances the Sensitivity of K562 Cells to Imatinib and Its Relative Mechanism

作　　者：林萍萍[1] 白雪佳[2] 岳志霞[1] 闫韶飞[2] 李宗文[2] 高超[1] 梅妍妍[1] 王凯玲[1] 李伟京[1] 丁卫[2] 李志刚[1] 

机构地区：[1]首都医科大学附属北京儿童医院血液病中心,北京100045 [2]首都医科大学基础医学院生物化学与分子生物学系,北京100069

出　　处：《中国实验血液学杂志》2012年第2期235-241,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金(编号30771062);北京市教育委员会科技发展计划(编号KM201010025001);北京市属高校人才强教(编号PHR201006111)

摘　　要：本研究探讨烟酰胺磷酸核糖转移酶(NAMPT)对K562细胞(人慢性髓系白血病细胞株)对伊马替尼敏感性的影响及其相关机制。在体外合成针对NAMPT基因的小干扰RNA(siRNA),转染K562细胞株。将转染后的K562细胞经1μmol/L伊马替尼作用48 h后,采用PI/Calcein染色方法测定细胞存活率;经不同浓度伊马替尼作用48 h后,采用MTS法测定细胞增殖活性,计算半数抑制浓度(IC50)。未经转染的K562细胞经1μmol/L伊马替尼作用3-48 h后,采用Western blot方法检测NAMPT表达的变化。通过对NCBI GEO中基因表达数据的挖掘分析及Western blot方法检测,探讨NAMPT-siRNA和伊马替尼对凋亡相关基因表达的影响。采用荧光素酶报告基因技术,研究NAMPT-siRNA、伊马替尼对NF-κB转录活性的影响。结果表明,1μmol/L伊马替尼作用于K562细胞48 h对NAMPT表达无明显影响。NAMPT-siRNA能够明显抑制K562细胞NAMPT的表达,再经1μmol/L伊马替尼作用后,NAMPT-siRNA干扰组细胞存活率明显降低(P<0.05),提示抑制NAMPT表达能增强K562细胞对伊马替尼的敏感性。不同浓度伊马替尼作用后,干扰组IC50值明显降低(P<0.05);拟合增殖曲线在0.01-0.1μmol/L伊马替尼浓度范围内斜率增大,提示在此范围内NAMPT-siRNA与伊马替尼的协同作用最强。基因表达谱分析及Western blot验证结果都显示,NAMPT-siRNA和伊马替尼处理均可下调NF-κB下游因子抗凋亡基因Bcl-2表达水平,两者同时作用时效果更明显。NAMPT-siRNA及伊马替尼均能降低NF-κB转录活性,两者同时作用效果更显著。结论:伊马替尼可能并非通过调节NAMPT表达影响细胞存活;抑制NAMPT表达能增强K562细胞对伊马替尼的敏感性,可能与抑制NF-κB及其下游因子Bcl-2有关。The aim of this study was to investigate the effect of suppression of nicotinamide phosphoribosyltransferase（NAMPT） expression on imatinib-sensitivity in chronic myelogenous leukemia（CML） cell line K562 and its mechanisms,NAMPT siRNA was synthesized and transfected into K562 cells.PI/Calcein staining technique was used to determine survival rate of transfected K562 cells at 48th hour after exposure to 1 μmol/L imatinib.MTS method was used to determine the proliferation changes of transfected K562 cell at 48th hour after exposure to different doses of imatinib,then half inhibitary concentration（IC50） was calculated.Expression of NAMPT at 3rd-48th hour after exposure to 1 μmol/L imatinib was determined by Western blot.To explore the effect of NAMPT-siRNA and imatinib on the expre ssion of apoptosis-related genes,the microarray data from NCBI GEO DataSets was analyzed,then the results were confirmed by Western blot.The luciferase reporter assay was used to determine the effect of NAMPT and imatinib on transcriptional activity of NF-κB transcription factors.The results showed that after exposure to 1 μmol/L imatinib for 3-48 h,there was no significant change of NAMPT expression in K562 cells. The expression of NAMPT could be effectively inhibited by the NAMPT-siRNA.After exposure to 1 μmol/L of imatinib for 48 h,the survival rate of NAMPT-siRNA interference group was lower than that of negative control group（P0.05）,indicating that suppression of NAMPT expression can increase the sensitivity of K562 cells to imatinib and enhance the killing effect of imatinib on K562 cells.The IC50 of imatinib in NAMPT-siRNA interference group was the lowest compared with that of control group（P0.05） after exposure to different concentrations of imatinib for 48 h,the fitted survival curves showed that the slope of NAMPT-siRNA interference group was the largest ranging between 0.01-0.1 μmol/L of imatinib. Data mining of expression profiling indicated that the anti-apoptotic factor Bcl-2 decreased in K562 cells

关 键 词：NAMPT 伊马替尼 NF-ΚB BCL-2 K562细胞 

分 类 号：R733.72[医药卫生—肿瘤] R979.1[医药卫生—临床医学]