多药耐药蛋白MRP4在耐ADM骨肉瘤细胞中的作用机制研究

【作者】 胡蓓蓓；

【导师】 林峰；

【作者基本信息】 苏州大学 ， 肿瘤学， 2013， 硕士

【摘要】 目的：阐明阿霉素（doxorubicin，ADM）对骨肉瘤细胞系MG63，骨肉瘤耐药细胞系MG63/DOX,以及原代培养骨肉瘤耐药细胞系GSF-0686细胞体外增殖抑制作用和多药耐药蛋白MRP4在细胞耐药中的作用。方法：1）CCK-8法检测ADM对MG63，MG63/DOX，GSF-0686细胞的增殖抑制作用。2）5μmol ADM分别作用于MG63，MG63/DOX，SF-0686三株细胞后，使用液相色谱-串联质谱法测定不同时间点三种细胞内外的ADM药物浓度。3）Western Blotting法与qPCR法检测、比较三种细胞株的MRP4的表达。4）加ADM刺激MG63和MG63/DOX细胞后，采用qPCR法分别检测两种细胞随加药时间和药物浓度变化时肿瘤耐药相关蛋白MRP4基因的表达。结果：1）ADM对MG63，MG63/DOX，GSF-0686的增殖均有抑制作用，但其对MG63/DOX和GSF-0686细胞的增殖抑制作用明显弱于MG63，有显著性差异（P＜0.05）。2）MG63/DOX和SF-0686细胞内ADM浓度明显低于MG63细胞内浓度，同时MG63/DOX和SF-0686细胞外ADM浓度明显高于MG63细胞外浓度。3）耐药细胞株MG63/DOX和SF-0686中MRP4的mRNA与蛋白表达量均高于亲本株MG63。4）ADM可下调MG63/DOX和MG63细胞MRP4的mRNA，但在耐药株中其下调作用弱于亲本株，差异均有统计学意义（p<0.05）。结论：（1）ADM对MG63/DOX和GSF-0686增殖的抑制作用较弱；（2）MG63/DOX细胞和SF-0686细胞对ADM耐药。3)MRP4可能通过表达上调促进细胞内药物外排参与MG63/DOX和SF-0686的耐药作用。更多还原

【Abstract】 Objective: To investigate the effects of doxorubicin (ADM) on proliferation ofosteosarcoma cell MG63, osteosarcoma multidrug resistant cell MG63/DOX, andprimary osteosarcoma resistant cell SF-0686, and the effect of multidrug resistanceprotein MRP4on drug resistant cells.Methods:1)CCK-8assay was used to investigate the proliferation of MG63,MG63/DOX and GSF-0686cells,2) The concentration of ADM in cells andextracellular fluid were detected at different time points after given the ADM withliquid chromatography-tandem mass spectrometry (LC/MS/MS).3)WesternBlotting and RT-PCR were used to detect the protein and gene expression of MRP4ofMG63, MG63/DOX and GSF-0686cells.y4)After given ADM, the gene expressionof MRP4was detected with time and the concentration using qPCR.Results:1)ADM inhibited the proliferation of MG63, MG63/DOX and GSF-0686cells, and the inhibitory effect on MG63/DOX and GSF-0686was lower than that onMG63(p＜0.05).2) The concentration of ADM in MG63/DOX and GSF-0686cellswas lower compared with MG63, and the concentration of extracellular fluid inMG63/DOX and GSF-0686cells was higher than that in MG63.3) The protein andgene expression of MRP4of MG63/DOX and GSF-0686cells were higher comparedwith MG63.4) The mRNA and protein expression of MG63/DOX and MG63cellswere down regulated after using ADM, but the effect on MG63was higher(p＜0.05).Conclusion:(1) The inhibitory effect of ADM on the proliferation of MG63/DOXand GSF-0686cells was weak,(2) MG63/DOX and SF-0686cells were resistant toADM,(3) multi-drug resistance associated protein MRP4plays an important role inmultidrug resistance of osteosarcoma.更多还原