CAJ | 학술논문

目的探讨鼠尾草酸(Canosic acid,CA)对人类白血病多药耐药(MDR)细胞系K562/A02细胞的逆转作用及机制.方法 MTT法测定CA作用前后K562/A02细胞对阿霉素(ADM)的敏感性.流式细胞术(FCM)和激光扫描共聚焦显微镜(LSCM)测定细胞内ADM的平均荧光强度,计算细胞内ADM浓度.半定量RT-PCR检测细胞mdr1 mRNA表达水平.采用流式细胞术和Western blot 检测细胞膜P糖蛋白(P-gp)表达.结果 CA可将ADM对K562/A02细胞的IC50值由16.31μg/ml降至1.35μg/ml,逆转倍数为12.08倍.流式细胞术检测结果表明CA可将K562/A02细胞内ADM的荧光强度由17.05提高到60.53(P<0.01).LSCM结果显示CA可恢复ADM在K562/A02细胞的细胞核和胞质中的弥散分布,并使细胞内ADM的浓度由4.9 Oμg/ml提高至15.4μg/ml.RT-PCR结果显示K562/A02细胞mdr1 mRNA水平明显高于K562细胞,CA处理后K562/A02细胞mdr1 mRNA水平明显降低(P<0.01).流式细胞术检测K562/A02细胞膜上P-gp的荧光强度在经CA处理后由44.40降至22.80(P<0.05).Western blot结果显示CA处理后的K562/A02细胞膜上P-gp的表达明显降低.结论在体外,CA可有效逆转人白血病细胞K562/A02的MDR,其逆转耐药的机制可能与P-gp蛋白表达下调并抑制其功能有关.
목적 탐토서미초산(Canosic acid,CA)대인류백혈병다약내약(MDR)세포계K562/A02세포적역전작용급궤제.방법 MTT법측정CA작용전후K562/A02세포대아매소(ADM)적민감성.류식세포술(FCM)화격광소묘공취초현미경(LSCM)측정세포내ADM적평균형광강도,계산세포내ADM농도.반정량RT-PCR검측세포mdr1 mRNA표체수평.채용류식세포술화Western blot 검측세포막P당단백(P-gp)표체.결과 CA가장ADM대K562/A02세포적IC50치유16.31μg/ml강지1.35μg/ml,역전배수위12.08배.류식세포술검측결과표명CA가장K562/A02세포내ADM적형광강도유17.05제고도60.53(P<0.01).LSCM결과현시CA가회복ADM재K562/A02세포적세포핵화포질중적미산분포,병사세포내ADM적농도유4.9 Oμg/ml제고지15.4μg/ml.RT-PCR결과현시K562/A02세포mdr1 mRNA수평명현고우K562세포,CA처리후K562/A02세포mdr1 mRNA수평명현강저(P<0.01).류식세포술검측K562/A02세포막상P-gp적형광강도재경CA처리후유44.40강지22.80(P<0.05).Western blot결과현시CA처리후적K562/A02세포막상P-gp적표체명현강저.결론 재체외,CA가유효역전인백혈병세포K562/A02적MDR,기역전내약적궤제가능여P-gp단백표체하조병억제기공능유관.
Objective To investigate the effects of carnosic acid(CA)on reversal of the muhidrug resistance(MDR)of human leukemia cell line K562/A02 and its mechanism.Methods MTT assay was used to determine the sensitivity of K562/A02 cells to adriamycin(ADM)pre-and post-treated with CA.Flow cytometry(FCM)and laser scanning confocal microscopy(LSCM)were used to measure intracellular fluorescence intensity and concentration of ADM respectively.The expression level of mdr1 was detected by semi-quantitative RT-PCR.P-glycoprotein(P-gp)expression was detected by FCM and Western blot.Resuits CA decreased,IC50 of ADM in K562/A02 cells from 16.31 μg/mL to 1.35μg/mL,being a 12.08fold decrease.The intracellular ADM fluorescence intensity of K562/A02 was increased from 17.05 t0 60.53after treated with CA(P<0.01).In living K562/A02 ceils,after treated with CA,the diffuse distribution of intracellular ADM was recovered in both nuclear and cytoplasm,and the concentration of intracellular ADM increased from 4.93μg/mL to 15.43μg/mL.RT-PCR assay showed that CA inhibited the expressions of mdr1 mRNA in K562/A02 cells(P<0.01).Mean fluorescence intensity of P-gp detected by FCM in CA-treated K562/A02 was decreased to 22.80 as compared with that in untreated K562/A02 cells(44.40,P<0.05).Conclusion CA can reverse the MDR of K562/A02 cells in vitro.The mechanism may be associated with down-regulation of mdr1 and inhibition of P-gp function.