白血病耐药细胞中葡萄糖神经酰胺合成酶对P-糖蛋白药物泵出功能的影响  

Influence of Glucosylceramide Synthase on P-glycoprotein Drug-efflux Function in K562/AO2 Cell Line

作　　者：张杨杨[1] 谢可鸣[1] 李玉玲[1] 穆会君[2] 殷莹[2] 张滨[2] 谢平[1,2] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院病理学与病理生理学系,江苏苏州215123 [2]无锡市人民医院中心实验室,江苏无锡214023

出　　处：《苏州大学学报（医学版）》2011年第4期605-608,612,共5页Suzhou University Journal of Medical Science

摘　　要：目的探讨人白血病多药耐药细胞株K562/AO2中葡萄糖神经酰胺合酶(GCS)对P-糖蛋白(P-gp)泵出功能的影响,以便进一步研究GCS在白血病细胞耐药形成中的作用机制。方法采用RNA干扰技术分别靶向干扰K562/AO2中的GCS和多药耐药基因1(MDR1),并通过荧光定量PCR检测小干扰RNA(siRNA)的干扰效果;用流式细胞术检测细胞内罗丹明123(rh123)的滞留量,以rh123的平均荧光强度(MFI)反映P-gp蛋白的泵出功能。结果 GCSsiRNA和MDR1siRNA对各自靶基因的抑制率分别是(68±5.72)%和(75.3±2.62)%;转染siRNA 48 h后,GCS干扰组MFI为255.75±76.1,MDR1干扰组MFI为357.25±41.57,分别是阴性干扰组的3.3倍和4.6倍。结论特异性的沉默GCS基因可以降低P-gp的泵出功能,提示GCS可通过协同P-gp的药物泵出功能参与白血病细胞的耐药形成过程。Objective To explore the influence on P-gp drug-efflux by glucosylceramide synthase（GCS）,study the molecular mechanism of GCS inducing drug-resistance in K562/AO2 leukemia cell line.Methods Applying RNA interfering（RNAi） technique GCS or MDR1 gene in K562/AO2 cells was silenced,and real-time PCR was used to detect the transfection effect.The mean fluorescence intensity（MFI） which represent intracellular rhodamine 123 retention（reflect P-gp function） was analyzed by flow cytometric.Results The expression level of GCSmRNA was inhibited by（68±5.72）% after GCSsiRNA was transfected,meanwhile,the expression of MDR1mRNA was also inhibited by（75.3±2.62）% after MDR1siRNA was transfected.In the GCS or MDR1 RNA interfering group,rhodamine123 retention significantly increased compared with that in the negative control group after RNAi for 48 hours.Conclusion Inhibiting GCS gene can decrease P-gp efflux function,which suggests that GCS may contribute to drug-resistance of human leuke-mia cell by cooperating with P-gp.

关 键 词：葡萄糖神经酰胺合成酶 P-糖蛋白 罗丹明123 RNA干扰 白血病 平均荧光强度 

分 类 号：R733.7[医药卫生—肿瘤]