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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 whether dimethyl amilo-ride (DMA), a specific inhibitor of Na + / H + exchanger-1 (NHE-1), can reverse drug- Multidrug Resistance of HL-60 / ADM and Its Possible Mechanism. Methods Adriamycin-induced multidrug resistance (MDR) HL-60 / ADM cell line was used as the research object. Chemosensitivity was detected by MTT assay and intracellular daunorubicin accumulation was detected by flow cytometry The mRNA expressions of mrp, bcl-2 and bax were detected by RT-PCR. The expression of multidrug resistance-associated protein (MRP) in cell membrane was detected by immunocytochemistry. The apoptosis of cells in situ was detected by TUNEL. Results IC50 values of ADM, MIT, VCR and HAR chemotherapeutic drugs in HL-60 / ADM cells were (839.5 ± 45.2), (321.2 ± 28.9) and (70.5 ± 15.5) (65.2 ± 20.7) ng / ml decreased to (180.5 ± 38.9), (76.4 ± 12.0), (30.2 ± 7.5) and (31.7 ± 7.2) ng / ml, The fluorescence intensity of DNR accumulation in HL-60 / ADM cells increased from 33.56 to 48.74 and 70.10 before the treatment, the expression of mrp mRNA, MRP protein and bcl-2 mRNA and the expression of bax mRNA in HL-60 / ADM cells increased significantly High apoptotic rates of HL-60 / ADM cells treated with 5μg / ml ADM were significantly increased after co-cultured with 100μmol / L DMA for 24h. Conclusions DMA can reverse the MDR of HL-60 / ADM cells, and its mechanism may be related to the down-regulation of MRP expression and the promotion of the apoptosis pathway of bcl-2 / bax.