转mdrl基因诱导cik细胞耐药并保持肿瘤杀伤活性

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1、中华血液学杂志2003年12月第24卷第12期ChinJHematol，December2003，Vol24.No.12转mdrl基因诱导CIK细胞耐药并保持肿瘤杀伤活性杨永红李惠芳石永进王逸群张英王奕嘉朱平[摘要]目的将多药耐药基因(mdr1)转入细胞因子诱导的杀伤(cytokine-inducedkiller，CIK)细胞，观察其是否产生耐药性并且保持肿瘤杀伤活性。方法用IFN-γ，CD3单抗，IL-2，IL-1等细胞因子体外诱导外周血单个核细胞获得CIK细胞。采用电穿孔方法将mdr1基因表达质粒pHam

2、dr，转入CⅨ细胞。转染后72h提取细胞总RNA，DNA酶消化残留质粒DNA后进行RT-PCR，鉴定mdr1基冈表达；流式细胞仪检测细胞膜耐药蛋白(P-gp)表达。四唑蓝比色法(MTT)检测转基因后CIK细胞对阿霉素和秋水仙碱的耐药性；同时检测转染前后CIK细胞对MCF7细胞(人类乳腺癌细胞系)的杀伤活性变化。结果转染mdr1后的CIK细胞mdr1mRNA阳性，P-gp阳性的CIK细胞为21％～37％(平均27％)。转染后CIK细胞获得了多药耐药性，对阿霉素的IC50值较转染前升高了22．3~45．8倍，对秋

3、水仙碱的IC50值升高了6．7～11．35倍。转染前后CIK细胞对MCF7肿瘤细胞的杀伤活性无明显变化(P>0．05)。结论CIK细胞转入mdrl基因后获得了多药耐药性，转染后的CIK细胞仍然保持原有的肿瘤细胞杀伤活性。[关键词]基因，mdr1；抗药性，多药；杀伤细胞，细胞因子诱导；基因转染CIKcellsacquiredmultidrugresistanceandmaintainedcytotoxicaetivitytotumorcellsaftermdrlgenetransfectionYalGYong-

4、hong，LIHui-fang，SHIYong-jin，WANGYi-qun，ZHANGYing，WANGYi-jia，ZHUPing.BeijngUnivensityFinstHospital，Beijing100034，China[Abstract]ObjectiveToinvestigatewhetherthecytokine-inducedkilier(CIK)cellscouldacquiremultidrugresistanceandmaintaintheoriginalcytotoxicacti

5、vityaftermultidrugresistance(mdr1)genestransfection.MethodsCIKcellsweregeneratedfromperipheralbloodculturedwithIFN-γ，CD3monoclonalantibody，IL-2，IL-1andtransfectedwithaplasmid(pHamdr)containinghumanmdrlgeneviaelectroporation，RT-PCRmethodwasusedtoassaymRNAexp

6、ressionofmdrlgeneintransfectecCIKcells，flowcytometrywithanti-P-gpmonoclonalantibodytodetectP-glycoprotein(P-gp)expressiononCIKcellsmembrane，andMTTassaytocompareboththemultidrugresistancetodoxorubicinandcolchicinesandcytotoxicactivitytohumanmammarycancelcell

7、lineMCF7betweentransfectedandnon-transfectedCIKcells．ResultmdrlexpressmnwasdetectedinthetransfectedCIKcells.TherewasastrongexpressionofP-gponthetransfectedCIKcellsandthepercentagesofP-gppositivecellswere21％～37％(average27％).TheIC50oftransfectedCIKcellstodoxo

8、rubicinwas22．3～45．8timesand6．7—11．35timestocolchicinesofthoseofnon-transtbctedCIKcells．ThecytotoxicactivitytoMCF7remainedunchanged(P>0．05)．ConclusionItdemonstratedthatCIKcellstransfectedwithmdrlgenevia