DNA损伤修复相关蛋白表达与辐射剂量关系的研究

摘要

IntroductionRapidandaccuratedeterminationofabsorbeddoseplaysanimportantroleinmanyfieldssuchasclassificationdiagnosis,clinicaltreatmentofradiationwounded,thepreciseradiationtherapyofcancerpatients,biologicaleffectstudiesinducedbyradiationandradiationhealthriskevaluationinspecialenvironment.Physicalmethodsofdoseevaluationforindividualexposurearedirect,accurateandauthoritative.However,inthesituationswithoutwearingpersonaldosimeterorwithoutinstallingdosemonitoringmeterson-site,thephysicalmethodsfordosedetectionaresometimesnotpossibleortimeconsumingbecauserebuildingthesceneofradiationneedstospendlotstimeandsometimesthesceneofradiationishardtoberebuild.Thus,insomespecialoremergencycircumstances,todetectpersonalexposuredoseonlyusingphysicalmethodsisunrealisticandnotpractical.Bio-analyticalmethodstoevaluatepersonnelexposuredoseshavemanyadvantagesandtheresearchesonbiodosimeterarethefuturedirectionofpersonalexposeddosesurvey.Howevertherearestillmanyscientificandtechnicalissuestoberesolvedinusingofbiologicalmethodsforrapidassessmentofpersonnelexposeddose.First,howtofilteroutaseriesofsensitiveandspecificbiomarkerswhichwellresponsetheradiationdose.Second,howtoestablishaaccurate,reliable,highlysensitiveandrapidanalysismethodbasedonthosebiomarkers.Withthedevelopmentofnewtheories,methodsandtechnologies,manynewbiologicalindicatorsingeneandproteinlevelsconstantlybediscoveredandreported.ForinstancetheresearchshownthatcontentandactivityoftheATMproteinwhichwasoneofDNAdamagerepair-relatedproteinshadagooddose-effectrelationshipwithradiationdose.ATMplaysanimportantrolenotonlyinDNArepairingbutalsointransportingDNAdouble-strandbreaksignal.ThecontentofATMactivationisconsideredtobedependentonthecontentofDNAdouble-strandbreak.WhentheDNAdouble-strandbreakoccursATMproteinitselfisphosphorylatedatthesametime.MeanwhilephosphorylatedATMgoestoactivateandregulateaseriesofDNArepair-relatedproteins,includingH2AX,RAD50,NBS1,Mre11,CHk1,2,c-AB1andmakesthemgatherintheDNAdamagesiteandpromotesDNArepair.Accordingtoreport,thesignalofactivationofATMproteincanbeseenat30minutesafter0.25Gyirradiation.FurthermoreahighbrilliantfluorescencefociofactivationofATMproteincanstillbedetectedat3hoursafter4Gyirradiation.Inthepast,peopleusuallyusedtheimmunofluorescencemethodtodetecttheATMactivation.AlthoughthismethodisrelativelystraightforwardinobservationofATMactivationincellsitcanonlydosemi-quantitativedetection.Thereforeitisdifficulttoestablishaccuraterelationshipbetweenamountofproteinactivationanddose-effect,especiallyinthesituationsoflowirradiationdoserangeandinnitialradiationstage.Accordingly,moresensitiveandspecificdetectionmethodsneedinresponsetofurtherstudywhichistherelationshipbetweenATMproteinexpressionoractivationandradiationdose.Currentlynano-materialshavebeenwidelyusedintheareasofDNAandproteinsensorsbecauseoftheirexcellentbiocompatibilityanduniqueopticalproperties.Inthisstudy,weusedauniquedesignednano-materialasprobewhichismodifiedbythechemicalorbiologicaltorealizesensitiveandspecificdetectiontoATMprotein...Thebasicstepsofthestudyareasfollows:first,thealdehydebeadsurfaceismodifiedbyATMmonoclonalantibodyandATMmulti-antibodytocapturetheantigeninthesamplesbyformingamonoclonalantibody-antigen-multi-antibodysandwichstructure.Andthennano-bio-probeisaddedintoreactionsystemtoachievesignalamplificationandoutput.Inthisstudythreenano-bio-probessuchasIgG-goldnanoparticle-DNAprobe,IgG-nano-Au-HRPprobeandIgG-carbonnanotubes-HRPprobeareinvolved.Amongthemasimple,stableandreliablemethodalsoisselectedtoachievetheobjective.Contents:SensitiveandreliablemethodsofATMproteindetectingarestudiedbasedonmodifiednano-materialsasprobescombinedwithmagneticbeadseparationandenzyme-linkedimmunosorbenttechnology.Accordingtothedifferentprobepreparationstheresearchisdividedintothreeparts:1.TheresearchofIgG-Nano-Au-HRPprobeforATMproteindetection2.TheresearchofmethodbasedonIgG-carbonnanotubes-HRPprobe.3.TheresearchofRCAmethodbasedontheIgG-goldnanoparticle-DNAprobe.MethodsandMeasures:1.MakingofgoldnanoparticlesandcarbonnanotubesprobeAdjustthepHofthegoldnanoparticlessolutionneartheisoelectricpointofaddingproteinandactivatethecarboxylofcarbonnanotubeusingEDCandNHSrespectely.ThenIgGat0.005mg/mlandHRPat1mg/mlwasaddedtopreparegoldnanoparticlesornano-carbontubemixtureandstirredovernightatroomtemperature.TheprobewasblockedwithBSAandwashedtoremoveanyfreeHRPandIgGandthepreparationswerestoredinrefrigeratorat4℃by0.5%caseinforuse.2.CheckofpreparedprobeTocheckifproteinswhichbandedonthesurfaceoftheprobepossessactivity,ATMpolyclonalantibodies-coatedmagneticbeads(blockedwith0.5%casein,0.1MPBS,PH7.4)werecoatedwithasolutionoftheprobe.ThesuccessfulconnectionofthetwoproteinsandbiologicalactivityoftheprobeweredetectedbythespecificcolorreactionbetweenIgGconnectedwithHRPontheprobeandpolyclonalantibodyofATMonthemagneticbeadswhenTMBwasaddedintosystem.3.DetectionoftargetproteinbytheprobeATMmonoclonalantibodies-coatedmagneticbeads(blockedwith0.5%casein,0.1MPBS,pH7.4)werecoatedwithasolutionofATMantigeninPBSat37℃for1h,washedfourtimesinPBS/0.25%Tween20.ATM-polyclonalantibodieswerethanaddedinto,at37℃for1h.Afterincubatingandwashing,probewasaddedin,andincubatedat37℃for1h.TheHRPontheprobewasdetectedbyTMB.4.DataanalysisSoftwareSPSS11.0wasUsedtoanalysethedata.One-dimensionalanalysisofvarianceforsignificanttest(asP<0.05forsignificantdifference).Thechartsproducedusingorigi8.0software.Thethresholdofdetectinglimitationwasidentifiedasthemeanofthenegativecontrolplusthreetimesstandarddeviationofthenegativecontrol.Thesignalishigherthantheconcentrationofthisvalueshallbetheminimumdetectionlimitofthemethod.Method-specificcanbeobtainedfromthedifferencebetweentheexperimentalgroupandtheembodiment.ResultsSWNTimmunosensorscanaccuratelydetecttheATMinstanderedsampleswithveryhighsensitivityandselectivity.Further,theimmunosensorshaveaverygoodreproducibility,asdemonstratedbydevice-to-devicestandarddeviationswithbothAb2-CNT-HRPbioconjugatesandconventionalsingle-labelHRP-Ab2.ThebestsensitivityisobtainedusingtheAb2-CNT-HRPbioconjugateswithhighenzymelabel/Ab2ratiosforsignalamplification.PreliminaryConclusionsPresentmethodsforthedetectionoftraceproteinhavetheirownnewideasandstrengths.Inthisresearchapplicationofcarbonnanotubeprobeintraceproteindetectingshowsacertainstabilityandcharacterofsignalamplificationcomparedwithothermethods.ThismethodguaranteednotincreasingtheexperimentalprocedureslikeRCAmethodbasedontheIgG-goldnanoparticle-DNAprobe,IgG-Nano-Au-HRPprobeandtheclassicalELISAmethoddidandkeepingtheadvantageinheritingfromtheelectrochemicalsystem.UsingthiskindofprobetacticswesuccessfullyachievedthespecificandsensitivedetectionofATMinanoptimizingsystemwhichdetectionlimitwaslower1pg/ml.

关键词