流式细胞术检测肿瘤耐药细胞中P-糖蛋白细胞前处理方法的优化

Optimization of Cell Pretreatment Methods for Detecting P-gp of MDR Cells by Flow Cytometry

目的:优化流式细胞术(FCM)检测肿瘤耐药细胞中P-糖蛋白(P-gp)的细胞前处理方法,提高细胞中P-gp的检出率。方法:采用抗原决定簇在细胞膜内的单抗JSB-1,以阳性百分比为优化指标,耐阿霉素人乳腺癌细胞系MCF-7/A为模型细胞,对FACSTM透化溶液的有无、FACSTM透化溶液的浓度、FACSTM透化溶液处理细胞的时间、鼠抗JSB-1抗体的孵育时间、羊抗鼠IgG-FITC(GAMIF)抗体的孵育时间、JSB-1抗体工作液的保存条件、以及细胞数量进行优化。结果:上述细胞处理方法对MCF-7/A的阳性百分比均有不同程度的影响;相对最佳的细胞处理方法为:5×105-20×105个细胞用FACSTM透化溶液的6倍稀释液处理10min,37℃下与JSB-1抗体孵育75min、与GAMIF抗体孵育30min,JSB-1抗体工作液于4℃保存或新鲜配制。结论:对FCM检测前的细胞处理方法进行优化,能显著提高肿瘤耐药细胞中P-gp的检出率,因而更能真实、客观地反映肿瘤耐药细胞的P-gp表达水平及耐药程度。

Objective： To optimize the cell pretreatment methods for detecting P-gp of MDR cells by flow cytometry （FCM） and increasing the detection rate of P-gp. Methods： By using JSB-1 with intramembranous antigen determinant as antibody, the positive rate as optimization index and MCF-7/A as model cells, cell pretreatment methods, such as the presence or absence of FACS^TM permeabilizing solution, the concentration of FACS^TMpermeabilizing solution, the time treating cells with FACS^TM permeabilizing solution, the time incubating cells with JSB-1 working fluid, the time incubating cells with goat-ani-mouse IgG-FITC （GAMIF） solution, and the conditions for keeping JSB-1 working fluid and cell numbers, were optimized. Results： These cell pretreatment methods influenced on the positive rate of MCF-7/A to some extent. The relative optimization methods were as follows： cells numbered from 5 × 10^5 to 20 × 10^5 were incubated with the six-time.diluted solution of FACS^TM permeabilizing solution for 10 min, andthen incubated with JSB-1 working fluid for 75 min and GAMIF for 30 min, respectively, at 37℃ ; in addition, JSB-1 working fluid was conserved under 4℃or was prepared freshly. Conclusion： By optimizing the cell pretreatment methods, the detection rate of P-gp of MDR cells could be increased significantly. And thus, the expression level of P-gp and the resistance degree of MDR cells could be revealed objectively by FCM.