鼠源乳腺癌多药耐药细胞株的建立及耐药机制的初步研究.docx

鼠源乳腺癌多药耐药细胞株的建立及耐药机制的初步研究[摘要]目的构建鼠源性乳腺癌5-氟尿嘧啶（5-Fu）耐药细胞株为研究乳腺癌耐药与体内免疫微环境的关系提供细胞模型。方法通过体外以浓度递增的方法诱导小鼠乳腺癌4T1细胞对5-Fu产生耐药MTS法确定其耐药性平板克隆检测其增殖活性实时定量和半定量PCR检测其中5-Fu代谢相关酶TS、MTHFR、TK、OPRT、DPD及药泵蛋白MDR1、MRP1的表达通过流式细胞术检测其周期及CD44+CD24-干细胞亚群的比例。结果耐药细胞4T1/5-Fu与4T1细胞相比4T1/5-Fu细胞对5-Fu、吉西他滨和顺铂耐药的耐药指数（RI）明显升高；5-Fu合成代谢相关酶TK、OPRT表达明显降低（P<0.05）代谢抑制性相关酶TS、MTHFR明显升高（P<0.05）药泵蛋白MDR1、MRP1表达明显升高（P<0.05）；流式细胞术结果显示CD44表达明显升高肿瘤干细胞群增多。结论成功构建小鼠乳腺癌多药耐药细胞株耐药发生可能与5-Fu代谢酶类、药泵蛋白表达以及干细胞比例改变相关。[关键词]肿瘤耐药；4T1细胞；5-氟尿嘧啶；肿瘤干细胞[中图分类号]R329.24[文献标识码]A[文章编号]1673-7210（2016）04（b）-0009-06[Abstract]ObjectiveToconstruct5-fluorouracil（5-Fu）resistantmurinebreastcancercelllineinordertoprovidecellmodelforstudyofrelationshipbetweenbreastcancermultidrugresistantandimmunemicroenvironmentinvitro.Methods4T1cellswereexposedinstepwiseescalatingconcentrationof5-Futodeveloptheresistantcellline4T1/5-Fu.Andthechemosensitivityandproliferationof4T1/5-FuweredeterminedbyMTSandcolonyformingexperimentsrespectively.Furthermorereal-timeRT-PCRandsemiquantitativePCRwereusedtomeasureexpressionlevelsof5-FurelatedgenessuchasTSMTHFRTKOPRTDPDMDR1MRP1.InadditioncellcycleandtheproportionofCD44+CD24-stemcellsubsetswereevaluatedbyflowcytometry.ResultsComparingto4T1cellstheresistanceindexof4T1/5-Fucellsto5-FuGEMcDDPwasincreased.Theresultsshowedthatcomparedwith4T1cellstheexpressionlevelsofTKandOPRTweresignificantlydecreased（P<0.05）whileTSMTHFRMDR1andMRP1wereremarkablyincreasedin4T1/5-Fucells（P<0.05）.FlowcytometryassayshowedthatthepropotionoftumorstemcellsCD44+CD24-wasevidentlyincreasedin4T1/5-Fucellsthanthatof4T1cells.ConclusionThisarticlehassuccessfullyconstructedmultidrugresistantcellline4T1/5-Fuwhoseresistancemaybeassociatedwithup-regulationofthe5-Fumetabolicenzymesdrugpumpproteinsexpressiona