NF-κB信号通路的激活对白血病细胞K562／A02多药耐药的影响

Effect of NF-κB signaling pathway on the formation of multidrug resistance in K562/A02 cells

目的研究K562细胞株及其耐药细胞株K562／A02 NF-κB活性的差异性表达，探讨白血病多药耐药（MDR）发病机制。方法用MTT法检测K562／A02的耐药倍数，观察细胞生长的形态学变化，PI单染法检测K562／S、K562／A02细胞周期分布，并用RT—PCR方法检测mRNA的表达、流式细胞仪检测P糖蛋白（P-gp）表达及其功能，Western blotting方法检测细胞核NF—κB p65表达，比较K562与K562／A02白血病耐药细胞株之间的差异性。结果与K562细胞不同，耐药细胞株K562／A02细胞生长特性发生改变，呈半贴壁生长，与K562／S细胞相比，K562／A02细胞的G0／G1期、S期比例增高，G2／M期细胞比例减低，差异有统计学意义（P〈0．05），凋亡细胞比例差异无统计学意义（P〉0．05），且检测到mdr1 mRNA及细胞膜P-gP表达增高以及细胞核内NF-κB p65表达明显增加。结论NF-κB信号通路的激活即NF-κB p65细胞核内转位可能参与了白血病MDR的形成。

Objective To explore the different expression of NF-κB in both K562 and its multidrug resistant cell line K562/A02 and discuss the mechanism of multidrug resistance（MDR）. Methods To detect the growing feature of the cells. Flow cytometry was used to analys the difference between the distribution profile of K562/S and K562/A02 cell. MTT colorimetry was used to determine the cytotoxic effect of adramycin, and expression of mdr1 gene was detected by semi-quantitative reverse transcriptase poly-merase chain reaction （RT-PCR） in K562 and K562/A02 cells. FACS was used to determine the expression and function of glycoprotein （P-gp） on the cell membrane. Western blotting was used to determine the NF-κB p65 protein in nucleus. Results There was a difference between K562 and K562/A02 cells growed in a half-adherent way rather than suspending ones, there were increases in the percentage number of cells at G0/G1 and S phases（P 〈0.05）. This was mirrored by a decreasing number of cells within the G2/M phase（P 〈0.05）. But there was no difference in apoptosis rate（P 〉0.05）. mdrl mRNA was detected in K562/A02 cells, in which the expression P-gp was much higher [（94.17±0.89）%：（1.41±0.491）%]. NF-κB p65 protein in nucleus was overexpressed in K562/A02 cells. Conclusion The activation of NF-κB signaling pathway may attribute to the formation of MDR in K562/A02 cells.