荧光实时定量PCR检测线粒体DNA氧化损伤方法初探

[7]EddyAA,FogoAB.PlasminogenActivatorInhibitor21inChronicKidneyDisease:EvidenceandMechanismsofAction[J].JAmSocNephrol,2006,17(11):2999.[8]FujisawaG,OkadaK,MutoS,etal.Spironolactonepreventsearlyrenalinjuryinstreptozotocin2induceddiabeticrats[J].KidneyInt,2004,66(4):1493.[9]MatsumotoS,TakebayashiK,AsoY.Theeffectofspironolactoneoncircu2latingadipocytokinesinpatientswithtype2diabetesmellituscomplicatedbydiabeticnephropathy[J].Metabolism,2006,55(12):1645.[10]GloriosoN,FilighedduF,ParpagliaPP,etal.11beta2Hydroxysteroidde2hydrogenasetype2activityisassociatedwithleftventricularmassines2sentialhypertension[J].EurHeartJ,2005,26(5):498.(:2009-03-20):(2008ZX10001-007);(30870853)3:1007-4287(2010)03-0320-05PCRDNA,,,,,,3(,100069):DNA,PCRDNA(MBL)DNA,8-DNA(OGG1)8-(82oxoG),PCROGG1DNADNA,OGG1;OGG1DNAPCR,DNACOCtOGG1,PCRDNA:DNA;;PCR:R39211:ADeterminationofoxidativemitochondrialDNAdamagewithReal2TimePCRSUNYu,ZHANGHong2hai,LIUYa2li,etal.(ResearchlaboratoryofSTDandAIDS,BeijingYouAnHospital,CapitalUniversityofMedicalScience,Beijing100069,China)Abstract:ObjectiveToconstructthemodelofoxidativeDNAdamageinvitro,usingreal2timePCRtomeasureoxidativemito2chondrialDNAdamage.MethodsTheplasmidwastreatedinvitrowithMethylenebluepluslight(MBL)toconstructthemodelofoxidativeDNAdamage.TheoxidativeDNAwassubjectedto82oxoguanineDNAglycosydase(OGG1)digestiontoconfirmtheforma2tionof82oxoguanine(82oxoG).Real2TimePCRwasusedtomeasuredifferentpercentofsamplesdigestedbyOGG1.ResultsAsuc2cessfulmodelofoxidativeDNAdamagehadbeenconstructed;OGG1candigestspecificoxidativedamagesite.ThestrongcorrelationwasfoundbetweenthepercentofsamplesdigestedbyOGG1andaveragecyclesamplifiedbyReal2TimePCR.ConclusionOGG1madebyourselveshadgoodbiologicactivity,andReal2TimePCRcouldassaytheoxidativeDNAdamagewithit.Keywords:mitochondrialDNA;oxidativedamage;RealTimePCR(ChinJLabDiagn,2010,14:0320)(mitochondrion,mt),,(reactiveoxygenspecies,ROS)ROS,DNA,DNA,8,8-(82oxoguanine,82oxoG),,DNA,[1]82oxoG:-(HPLC2ECD)(ELISA),32P023ChinJLabDiagn,March,2010,Vol14,No.3(GC2MS)(HPCE)PY[2],,(Methy2lenebluepluslight,MBL)DNA,82oxoG8-DNA(82oxoguanineDNAglycosydase,OGG1),PCR,,11.1(HepG2)pSEAP22Con2trolVectorPromega,pTrcHisVectorInvitrogenBamH(IPTG)TaKaRa(Methyleneblue,Mb)SigmaDMEMGIBCONi2NTAArgaroseQIAampDNAMiniKitQIAquickGelExtractionKitQIAGENPCRInvitrogenABI7900HTRT2PCR,SequenceDetectionSystem(SDS)(ABI,2.3)SDSRQManager(ABI,1.2)ThermoScientific8000CO2(Thermo)SPSS11.51.2DNA10%DMEM375%CO2HepG2,QIAampDNAMiniKitDNA1.3DNA[3]1.3.1pSEAP22ControlVectorHepG2DNA1%TAE-,pSEAP22ControlVectorDNA(ddH2O),200g/ml1.3.2100MMB125ddH2O,pSEAP22ControlVectorDNA,ddH2OpSEAP22ControlVector1.3.3,10018cm,301.3.4QIAquickGelExtractionKitDNA1.3.5pSEAP22ControlVectorBamH:10KBuffer20l,BamH5l,MBLpSEAP22ControlVector75l,ddH2O200l;pSEAP22ControlVector,372QIAquickGelExtractionKit,1%TAE-1.4OGG1[4]OGG1cDNApTrcHisvector,,IPTG,Ni2NTAAgaroseOGG1,1.5OGG11.5.1OGG1pSEAP22ControlVector:10OGG1Buffer1l,OGG10.5l,MBLpSEAP22ControlVector2l,ddH2O10l,pSEAP22ControlVector,372,1%TAE21.5.2OGG1DNA:10OGG1Buffer8l,OGG14l,MBLDNA16l(200g/ml),ddH2O80l,372:MBLDNA16l(200g/ml),ddH2O80l,3721.6PCR1.6.1(C(cytochromeCoxidase,CO)ABIPrimerExpress3.0:CO:52CCACCAATGGTACTGAACCTACGAG23,52TGTGGTTTGCTCCACAGATTTCAG23,52FAM2CAATTCCCGGACGTCTAAACCAAACCACTTTC2TAMRA231.6.2PCROGG1MBLDNA10123201031430,82,64,55,46,28,010PCR7DNA,OGG1DNA100%80%60%50%40%20%0MBLOGG1DNADNA100,82,64,55,46,28,010PCR7DNA,MBLOGG1DNA100%80%60%50%40%20%01.6.3PCRTaqmanUniversalPCRMasterMix(2)10l,0.4l(10M),0.4l(5M),DNA0.8l,ddH2O20lddH2O,3:9510min,9515s6060s,401.7,P0.05SPSS11.522.1DNApSEAP22ControlVectorDNA1%TAE2,,55ng/l45ng/l(1)M:DNAMarker;1:pSEAP22ControlVector,2:DNA1pSEAP22ControlVectorDNABamHpSEAP22ControlVectorMBLpSEAP22ControlVector,BamHDNA,(2)M:DNAMarker;1:MBLpSEAP22ControlVector,2:pSEAP22ControlVector2MBLpSEAP22ControlVector2.2pSEAP22ControlVectorOGG1pSEAP22ControlVectorpSEAP22Con2trolVectorMBLBamH,82oxoGDNA,82oxoGOGG1,buffer1%TAE-(3):OGG1MBL,DNA(4)2.3PCR,OGG1DNA100%80%60%50%40%20%0,(Ct)20.55919.33219.08619.04818174217123616.581Bivariate,OGG1DNA,r0197,P0.01(5),MBLOGG1DNA100%80%60%50%40%20%0,Ct16.58216.63816.81416.65116.74516.62416.685Bivariate,223ChinJLabDiagn,March,2010,Vol14,No.33MBLpSEAP22ControlVectorM:DNAMarker;:MBLpSEAP22Con2trolVector(OGG1OGG1),pSEAP22Con2trolVector(OGG1OGG1)4MBLpSEAP22ControlVectorOGG13DNADNA,16569bp,22tRNA2rRNA13DNA,,,,DNA10[5]DNA,,DNA,DNA82oxoG,82oxoA,30,[2]DNA5OGG1DNA82oxoGDNA,DNA82oxoG,DNAG:C2T:A,DNA[6],82oxoGhOGG1,DNAC82oxoG,(BaseExcisionRepair,BER)G:C,[7]MBLDNA,DNA82oxoG,OGG182oxoG,(3)2,MBLMBLOGG1,OGG1,MBL,DNA,MBLOGG1,,DNA,32320103143OGG1,[8,9],DNA,DNA,DNA2-3/10kb[10],OGG1DNA,MBLDNA,DNA,PCR,Ct,DNA82oxoG,DNA,CO[11],DNACO,OGG1MBLDNA,PCR,,DNA,Ct,OGG1,PCRDNA,DNA,MBL82oxoG,DNA100%PCRPCRMBL,PCR,MBLOGG1DNADNAPCR,,MBLDNA,CtPCR,,,,PCR82oxoGOGG1,OGG1PCRDNADNA,PCRDNA,,,,CO200bp,,,,:(1981-),,,:[1]ShigenagaMK,AmesBN.Assaysfor82hydroxy222deoxyguanosine:abiomarkerofinvivooxidativeDNAdamage[J].FreeRadicBiolMed,1991,10(324):211.[2]SinghR,TeichertF,VerschoyleRD,etal.Simultaneousdeterminationof82oxo222d