米非司酮逆转K562/A02细胞多药耐药的研究  

Study of mifepristone on reversing multidrug resistance of leukemic cell line K562/A02

作　　者：孔德晓[1] 陈春燕[1] 许复郁[1] 贾继辉[2] 

机构地区：[1]山东大学第二医院血液肿瘤科,济南250033 [2]山东大学实验畸形学教育部重点实验室

出　　处：《中华血液学杂志》2007年第8期555-559,共5页Chinese Journal of Hematology

基　　金：山东省科技攻关重点资助项目(2004GG2202107);山东省优秀中青年科学家奖励基金(2004BS02007);济南市科技青年明星计划资助项目(50114)

摘　　要：目的研究孕激素拮抗剂米非司酮对白血病多药耐药细胞 K562/A02的逆转作用及其机制。方法 MTT 法检测米非司酮作用72 h 后 K562/A02细胞增殖及其对阿霉素杀伤敏感性的变化;流式细胞术检测米非司酮作用前后 K562/A02细胞表面 P 糖蛋白的表达和细胞内柔红霉素的浓度;免疫组化法观察米非司酮作用前后 K562/A02细胞凋亡相关蛋白 bcl-2、Bax、caspase-3的表达;RT-PCR 检测米非司酮作用后对 K562/A02细胞内葡萄糖神经酰胺合成酶(GcS)mRNA 表达的影响。结果2.5、5.0和10.0μmol/L 米非司酮不抑制 K562/A02细胞的增殖,但上述浓度的米非司酮作用后K562/A02细胞对阿霉素的敏感性较前分别增强了1.68、4.17和10.71倍。K562/A02细胞表面 P 糖蛋白的表达为(49.03±5.32)%,10 μmol/L 米非司酮作用72 h 后降低到(28.60±2.13)%(P<0.01);K562/A02细胞内柔红霉素的浓度为(61.07±8.61)%,而10 μmol/L米非司酮作用后升高到(92.72±3.48)%(P<0.01)。经10 μmol/L 米非司酮作用后,bcl-2蛋白表达由(56±9)%降低到(37±6)%(P<0.05);Bax 蛋白由(40±5)%升高到(87±10)%(P<0.01);caspase-3蛋白则由(36±7)%升高到(89±6)%(P<0.01)。RT-PCR 结果显示 K562/A02细胞 GcS mRNA 的表达较K562细胞明显升高,10 μmol/L 米非司酮能明显降低 K562/A02细胞内 GcS mRNA 的表达。结论米非司酮可逆转白血病 K562/A02细胞的多药耐药,且具有剂量依赖性。10 μmol/L 米非司酮能明显逆转白血病 K562/A02细胞的多药耐药,其机制与降低 P 糖蛋白的水平,调节凋亡相关蛋白 bcl-2、Bax、caspase-3的表达,降低 GcS mRNA 有关。Objective To study whether progestogen antagonist mifepristone could reverse multidrug resistance of K562/A02 cells and its mechanisms. Methods MTF was used to study the proliferation of K562/A02 cells and sensitivity of K562/A02 cells to ADM after 72 hours treatment with mifepristone. Flow cytometry was used to assay the expression of P-glycoprotein and the mean fluorescent intensity of intracellular daunorubicin. The expressions of apoptosis related proteins （bcl-2 ,Bax and caspase-3 ） were assayed by immunohistochemistry and the glucosylceramide synthase mRNA expression by RT-PCR before and after mifepristone treatment. Results MTF assay revealed that 2.5, 5.0 and 10 μmol/L mefepristone did not affect the proliferation of K562/A02 cells, but enhanced the sensitivity of K562/A02 cells to ADM, by 1.68-, 4.17- and 10.71- fold increase, respectively. Expression of P-gp in K562/A02 cells was （49.03 ± 5.32 ）% , and was decreased to （28.60 ± 2.13 ） % （ P 〈 0.01 ） after 10 μmol/L mifepristone treatment for 72 hours. and intracelluar DNR accumulation in K562/A02 was （61.07 ± 8.61 ）% , and was increased to （92.72 ± 3.48 ） % （ P 〈0.01 ）. After 10 μmol/L mifepristone treatment, the expression of bcl-2 protein was decreased from （56 ± 9） % to （ 37 ± 6） % （ P 〈 0.05 ）, Bax and caspase-3 proteins was increased from （40 ± 5 ） % to （ 87 ± 10 ） % （ P 〈 0.01 ）, and from （ 36 ± 7 ） % to （89 ± 6 ） % （ P 〈 0. 01 ） respectively. RT-PCR analysis revealed that expression of glucosylceramide synthase mRNA was higher in K562/A02 than in K562 cells, whereas 10 μmol/L mifepristone significantly down-regulated its expression in K562/A02 ceils. Conclusion Mifepristone at 10 μmol/L could dose-dependently reverse the muhidrug resistance of K562/A02 cells. The possible mechanisms are related with decreasing the expression of P-gp, regulating the expression of apoptosis related proteins and decreasing the expression of glucosylceramide synthase.

关 键 词：米非司酮 细胞系 K562/A02 抗药性 多药 多药耐药蛋白 细胞凋亡 

分 类 号：R733.7[医药卫生—肿瘤]