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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 investigate the effect of glucosylceramide synthase (GCS) on the p-glycoprotein (P-gp) pumped-pump function in human leukemia multidrug-resistant cell line K562 / AO2 in order to further study the effect of GCS on the drug resistance of leukemic cells Mechanism. Methods The interference of GCS and multidrug resistance gene 1 (MDR1) in K562 / AO2 were detected by RNA interference technique. The interference effect of small interfering RNA (siRNA) was detected by fluorescence quantitative PCR. The intracellular Rhodamine 123 (rh123) retention, the average fluorescence intensity of rh123 (MFI) reflects the p-gp protein pumping function. Results The inhibitory rates of GCS siRNA and MDR1 siRNA on target genes were (68 ± 5.72)% and (75.3 ± 2.62)%, respectively. The MFI of GCS interference group was 255.75 ± 76.1 and the MFI of MDR1 interference group was 357.25 ± 41.57, respectively, 3.3 times and 4.6 times the negative interference group. Conclusion The specific silencing GCS gene can reduce the pump-out function of P-gp, suggesting that GCS can participate in the drug-resistance process of leukemia cells by cooperating with P-gp drug pumpout function.