NHE-1特异性抑制剂DMA逆转HL-60/ADM细胞多药耐药的实验研究  

Reversal of multi-drug resistance in HL-60/ADM cells by dimethyl amiloride in vitro

作　　者：常城[1] 孔佩艳[1] 陈幸华[1] 彭贤贵[1] 刘林[1] 刘红[1] 曾东风[1] 梁雪[1] 王庆余[1] 

机构地区：[1]第三军医大学新桥医院血液内科,重庆400037

出　　处：《第三军医大学学报》2007年第4期328-331,共4页Journal of Third Military Medical University

基　　金：第三军医大学新桥医院创新性基金(2001D068)~~

摘　　要：目的探讨钠/氢交换器-1(Na+/H+exchanger-1,NHE-1)特异性抑制剂二甲基氨氯吡咪(dimethyl amilo-ride,DMA)是否能在体外逆转耐药细胞株HL-60/ADM的多药耐药及其可能机制。方法以阿霉素诱导产生多药耐药(multidrug resistance,MDR)的HL-60/ADM细胞株为研究对象,用MTT法检测化疗药物敏感性,流式细胞仪检测细胞内柔红霉素积累量,RT-PCR法检测mrp、bcl-2、bax mRNA表达,免疫细胞化学法检测细胞膜多药耐药相关蛋白(MRP)表达,TUNEL法检测原位细胞凋亡。结果与50μmol/L DMA共同培养24h后,HL-60/ADM细胞对ADM、MIT、VCR、HAR4种化疗药物的IC50值分别由(839.5±45.2)、(321.2±28.9)、(70.5±15.5)、(65.2±20.7)ng/ml降低至(180.5±38.9)、(76.4±12.0)、(30.2±7.5)、(31.7±7.2)ng/ml;分别与50、100μmol/LDMA共培养24h后,HL-60/ADM细胞内DNR积累量荧光强度由作用前的33.56升高到48.74和70.10,细胞内mrp mRNA表达量、MRP蛋白表达量及bcl-2mRNA表达量明显降低,bax mRNA表达量明显升高;与100μmol/L DMA共同培养24h后,5μg/ml ADM作用下的HL-60/ADM细胞凋亡率明显增高。结论DMA可逆转HL-60/ADM细胞的MDR,其机制可能与下调MRP表达及促进bcl-2/bax凋亡途径有关。Objective To investigate the reversal and the possible mechanism of muhidrug-resistance （MDR） by dimethyl amiloride （ DMA）, a specific inhibitor of Na^＋/H^＋ exchanger-1 （ NHE-1 ）, in HL-60/ADM cell line in vitro. Methods After DMA treatment, the chemosensitivity of HL-60/ADM cells was studied by MTF assay. Intracellular concentration of daunorubicin （DNR） was determined by flow cytometry. Expression of mrp mRNA, bcl-2 mRNA, bax mRNA was assayed by semi-quantitive RT-PCR. MRP protein expression was detected immunohistochemically. Cell apoptosis was observed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL）. Results After treated with 50 μmol/L DMA for 24 h, IC50 of HL-60/ ADM cells to ADM, MIT, VCR and HAR decreased from （839.5 ± 45.2）, （321.2 ± 28.9 ）, （70.5 ± 15.5 ） , （65.2 ±20.7） ng/ml to （180.5 ±38.9） ,（76.4 ±12.0） ,（30.2 ±7.5） ,（31.7 ±7.2） ng/ml. After treated with 50 μmol/L or 100 μmol/L DMA for 24 h, the intracellular concentration of DNR in HL-60/ADM cells increased from 33.56 to 48.74 or 70.10, and expression of mrp mRNA, MRP protein and bcl-2 mRNA decreased significantly, but bax mRNA increased significantly. After treated with 100 μmol/L DMA for 24 h, the rate of apoptotic cells in HL-60/ADM cells treated with 5 μg/ml ADM increased significantly. Conclusion DMA could reverse MDR of HL-60/ADM cells in vitro, and the mechanisms were associated with decrease of MRP expression and induction of cell apoptosis through bcl-2/bax pathway.

关 键 词：二甲基氨氯吡咪 钠/氢交换蛋白-1 HL-60/ADM 多药耐药 逆转 

分 类 号：R730.53[医药卫生—肿瘤] R733.7[医药卫生—临床医学]