细胞酸化对P糖蛋白高表达耐药白血病细胞的影响  

Effect of intracellular acidification on drug resistance of leukemia cells with high P-glycoprotein expression

作　　者：李庆华[1] 芦颖[1] 金薇娜 蔺亚妮 胡蓉华 竺晓凡 王建祥 庞天翔 

机构地区：[1]中国医学科学院、北京协和医学院血液学研究所、血液病医院,实验血液学国家重点实验室,天津300020

出　　处：《中华血液学杂志》2009年第9期605-609,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金（30570358）;天津市自然科学基金（08JCZDJC19100）

摘　　要：目的探讨细胞酸化对患者原代白血病细胞P糖蛋白（P—glycoprotein，P—gp）和mdrl表达的影响，为逆转白血病多药耐药（MDR）提供新方法。方法采用实时定量RT—PCR技术检测mdrl mRNA表达水平的变化，筛选出P—gp高表达的患者白血病细胞。应用钠氢离子交换蛋白1特异性抑制剂Cariporide和高钾缓冲液对细胞进行酸化，采用MTT法观察细胞酸化对细胞活力的影响。应用激光共聚焦显微镜测定细胞内pH值（Intracellular pH，pHi）及细胞酸化对细胞内阿霉素累积的影响，应用流式细胞术检测细胞酸化对细胞中P—gp功能的影响，采用Western blot检测细胞酸化对细胞P—gp表达水平的影响。结果患者细胞pHi降低到7．0，酸化1h后mdrl mRNA表达量分别降至对照组的0．532±0．110，酸化3h后降至对照组的0．166±0．070；酸化3h后P—gp的蛋白水平降至对照组的0．560±0．090；细胞pHi 7．01h后细胞内罗丹明123（Rhodaminel 123，Rh123）累积由对照组的20．07±0．39明显增加至71．03±0．47，激光共聚焦显微镜观察结果也证实酸化增加细胞内阿霉素累积。结论细胞内酸化能够抑制患者细胞P—gp表达和功能。Objective To investigate the impact of intracellular acidification （IA） on drug resistance of leukemia cells with high P-glycoproteiu （P-gp） expression, and to provide a new method for the reversing of muhidrug resistance （MDR）. Methods Real-time PCR was used to determine the expression level of mdrl gene, and the leukemia cells with high P-gp expression were selected. The specific inhibitor of Na^＋/ H ^＋ exchanger 1 and the ＂high K ^＋ ＂ buffer were used to acidify the cells, and the confocal laser microscopy was used to determine the intracellular pH （ pHi ） and effect of IA on the accumulation of doxorubicin. The MTT method was used to determine the effect of IA on the cell viability. The flow cytometry was used to detect the effect of IA on the P-gp function, and Western blotting was used to determine the effect of IA on the expression of P-gp. Results The pHi was decreased to 7.0, and compared with that of control the mdrl mRNA expression was decreased to （53.2 ± 11.0） % after 1 h , and to （ 16.6 ±7.0） % after 3 h treatment. The P- gp expression was decreased to （56.0 ±9.0） % of the control after 3 h treatment. The accumulation of Rh123 was 71.03 ±0.47 at pHi 7.0, which was increased obviously as compared to the control group 20.07 ±0.39. The increased accumulation of doxorubicin was also observed by confocal laser microscopy. Conclusion The expression and function of P-gp on the patients cells are inhibited by IA.

关 键 词：Cafipofide 细胞酸化 P糖蛋白 抗药性 多药 

分 类 号：R686[医药卫生—骨科学]