汉防己甲素预防K562细胞获得性耐药机制的研究

Mechanisms of Preventive Effect of Tetrandrine on Acquired Multidrug Resistance in K562 Cells

本研究从MDR1基因转录调控和细胞凋亡两方面探讨汉防己甲素(TTD)预防K562细胞阿霉素(ADM)诱导性多药耐药(MDR)产生的作用机制。本实验分3组,第1组为K562细胞空白对照组;第2组用ADM作用K562细胞24小时诱导细胞耐药;第3组采用TTD预处理K562细胞24小时后,再用ADM诱导耐药。采用RT-PCR检测各组细胞转录因子c-Jun、YB-1及Survivin的mRNA表达水平;Western blot法检测c-Jun、YB-1的核内蛋白表达水平;流式细胞术检测细胞的凋亡程度。结果表明,0.6μg/ml阿霉素作用后K562细胞MDR1基因转录上调;与空白对照组(第1组)相比,ADM诱导的耐药细胞组(第2组)c-Jun mRNA和蛋白表达减低(p均<0.05),YB-1 mRNA和蛋白表达明显上调(p均<0.05);Survivin mRNA表达无明显差异(p>0.05);细胞凋亡率(8.31%)比空白对照组(0.75%)稍有增加;TTD预处理后再用ADM诱导组(第3组)与ADM作用组相比,c-Jun mRNA和蛋白表达增加(p均<0.05);YB-1 mRNA和蛋白表达下调(p均<0.05);Survivin mRNA表达明显减少(p<0.05),细胞凋亡率(97.2%)明显增加。结论:TTD预防白血病细胞耐药形成的机制可能与其下调YB-1基因和蛋白的表达、上调c-Jun基因和蛋白的表达,从而下调MDR1基因表达有关,另外,TTD与ADM联合作用还能抑制Survivin的表达,增加白血病细胞的凋亡。

This study was purposed to explore the mechanisms of preventive effect of tetrandrine（TTD） on doxorubicin（ADM）-induced multidrug resistance（MDR） in human leukemia cell line K562 from two aspects of the transcription control of MDR1 gene and cell apoptosis.The experiment was divided into 3 groups： group Ⅰ—blank control;group Ⅱ—ADM-induced drug-resistance;group Ⅲ—ADM-induced drug-resistance after pretreatment with TTD.Reverse transcription-PCR（RT-PCR） was used to detect the mRNA expression levels of c-Jun,YB-1 and Survivin genes.Western blot was used to determine the nuclear protein expressin levels of c-Jun and YB-1.Flow cytometry was used to assay the apoptosis of cells.The results showed that as compared with group Ⅰ,the expression levels of c-Jun mRNA and nuclear protein decreased（p〈0.05）,as well as the expression levels of YB-1 mRNA and nuclear protein increased in group Ⅱ（p〈0.05）.However,the expression of Survivin mRNA had no change（p〉0.05）;the apoptosis rate of cells was 8.31%.As compared with group Ⅱ,the expression levels of c-Jun mRNA and nuclear protein increased（p〈0.05）,expression levels of YB-1 mRNA and nuclear protein as well as Survivin mRNA decreased in group Ⅲ（p〈0.05）.The apotosis of cells was 97.2%.It is coucluded that TTD can inhibit the expression of YB-1 and up-regulate the expression of c-Jun,thus inhibit the expression of MDR1 gene.TTD can also inhibit the expression of Survivin and increase the apoptosis of cells induced by ADM.