溴化乙锭诱导无线粒体DNA乳腺癌T47D细胞系的建立与其细胞生物学特征和超微结构的改变

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1、AbstractBreastcancerisoneofthemostcommonmaliganciesinfemaleacrosstheworld．Inwesterncountries，breastcancerranksfirstamongallthefemalecancers；whileasinchina，althoughbreastgo．ricerisstilloflowincidencerateingeneral，ithasdramasticallyincreasedabout30％inthepast10years．Inadditio

2、n，inmostbigcitiessuchasBeijing，Shanghai&Tianjin，breastcancerhasalsojumpedtothefirstinthefemalemaligancyincidence—rankinglist．Inrecentyears，thecorrelationbetweenalterationsinmitochondrialDNA(mtDNA)andvarioussortsofsolidtumorsincludingbreasttumorhasbeenbroadlyinvestigated．in

3、ordertofurtherexplorethepotentialfunctionofmtDNAmutationintheetiologyandpathogenesisofbreasttumorandelucidatethemoleculargeneticmechanismofnucleo．mitochondrialinteraction，itisusefulandnecessarytoisolateaspecialcellmodelthatcompletelylacksmtDNA，denotedasmtDNA—lessorrho—zero

4、(Po)cellline．ThepocelllineallowsustomanipulatethemtDNAcomplementofacell，movemitochondriafromonecellularenvironmenttoanother,orintroducenewgenesintomitochondriaandcouldworkasallexcellentresearchplatformforbetterunderstandingthepotemialcontributionsofmitochondrialgenomeinhum

5、anmaligancies．TheprimaryobjectiveofourprojectistoestablishaviablebreastcancerT47DcellmutantdepletedofmtDNAbyapplyinglow-doseethidiunabromide(EtBr)，awell-knownmtDNAreplicationinhibitor,andthenexplorestheeffectsofdepletingmtDNAoncellularprocessesandultrastructureincomparisio

6、nwithitsparentalcellline．Therearemainlytwopartsinourresearch：PartI：Todeveloppocells．T47Dcellswerechronicallyexposedto50ng／mlEtBrinmediumsupplementedwithpyruvateanduridine．Afterapproximately32days．theEtBrtreatmentwasdiscontinuedandpocellswerereadyforfurtheridentification．In

7、ourT47Dpocells，DNAassays(southerndotblotsandreal—timePCR)confirmedtheabsenseofmtDNAandselectivepotestmediumanalysisverifieditsautrophicgrowthcharacteristicsforuridine．TheT47DpocellderivativecansteadilygrowanddivideinRPMI1640supplementedwitll15％FBS．sufficienturidineandpyruv

8、ate．3PanII：Byusingseveralmorphologicandproliferativeassays(inversemicroscope．MTTgrowthcur