胶质瘤细胞耐药性逆转的HPLC法研究

Reversal action research in drug-resistance of glioma cells by high performance liquid chromatography

目的 检测胶质瘤细胞中阿霉素 (ADM )的含量 ,探讨抗P 糖蛋白 (P GP)单克隆抗体对细胞耐药性的逆转效果。方法 将胶质瘤细胞分为敏感组、耐药组、耐药 +抗P GP单抗组及空白对照组 ,采用高效液相色谱 (HPLC)法测定细胞中ADM含量。色谱条件为LiChrosorbC18柱 (2 5 0mm× 4 .6mm ,10 μm) ;流动相为甲醇 乙腈 0 .0 1mol·L-1磷酸二氢胺 冰醋酸 (5 0 2 2 2 8 0 .5 ) ;流速为 1ml·min-1;激发波长为 4 95nm ,发射波长为 5 6 0nm。结果 测得细胞内ADM浓度在 0 .1～ 1.0mg·L-1范围内呈线性关系 ,相关系数为 0 .9983。ADM在各种细胞中的浓度分别为 空白对照组 (0 .4 96± 0 .0 15 )mg·L-1,敏感组 (0 .5 33± 0 .0 2 9)mg·L-1,耐药组 (0 .5 0 2± 0 .0 75 )mg·L-1,耐药 +抗P GP单抗组 (0 .5 77± 0 .0 86 )mg·L-1。结论 HPLC法是一种简便而准确地检测细胞中ADM的方法。结果表明 ,与正常对照组相比 ,耐药的胶质瘤细胞中ADM浓度降低 ,加抗P GP单抗逆转后的细胞中ADM浓度升高 ,提示抗P

Objective To determine the concentration of adriamycin(ADM) in glioma cells and the reversal action of monoclonal antibody(McAb) of P GP to drug resistance of glioma cell.Methods The glioma cells were divided into four groups: the controls, drug sensitive cells, drug resistant cells, drug resistant cells with McAb. High performance liquid chromatography(HPLC) was used to determinate the concentration of glioma cells. The cells in each group were chromatographed on a LiChrosorb C18 column(250 mm× 4.6 mm,10 μm) with a mobile phase consisting of methyl alcoholacetonitrile0.01 mol·L -1 ammonium dihydric phosphateacetic acid(5022280.5) at a flow rate of 1 ml·min -1 and detected by fluorescence detector at excitation and emission wavelengths of 495 and 560 nm respectively.Results A linearity was obtained from 0.1 to 1.0 mg of adriamycin per litre with a correlation coefficient of 0.9983. The concentrations of ADM in different groups of cells were (0.496±0.015) mg·L -1 (the controls), (0.533±0.029) mg·L -1 (drug sensitive), (0.502± 0.075 ) mg·L -1 (drug resistant) and (0.577±0.086) mg·L -1 (drug resistant with McAb) respectively. Conclusion HPLC is a convenient and exact method for determination of ADM in cells.The results show that the concentration of ADM in drug resistant cells is low while high in cells with McAb of P GP.It suggests that P GP McAb has a reversal action to drug resistance of glioma cell to adriamycin.