ape1在线粒体dna损伤修复中的作用与其线粒体定位机制的分析

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1、BecauseofacrucialroleincellactivityofmtDNA，oVer—expressinginjuryafteroxidatiVestressand1imitedDNArepairDNArepairenzymesinmitochondriarepresentsapotentialstrategyforpreventingeVenreVersingtheinitiationandprogressionofoxidative—relateddiseases．ManyrecentstudiesshowedthattargetingDNAglycos

2、ylaseinmitochondriaresultedinincreasedcellsurvivalafteroxidatiVestress．HoweVer，preViousover—expressionexperimentswithheterologousAPendonucleaseinmitochondriaproVidedconfUsingresults．HereinwetargetedatruncatedhumanAPE1proteinspecificallyinmitochondriaofhumanumbilicalveinendothelialceUs(H

3、UVECs)byaddingmitochondrialtargetingsequence(MTS)ofhumanmanganesesuperoxidedismutase(MnSOD)．ThenweinvestigateditseffectonmtDNArepaircapacityandceUsurViValafteroxidativestress．objectiVe1．ToinvestigatethesubcellulardistributionalterationofAPE1afteroxidatiVestressinducedbyIRandhydrogenpero

4、xide．2．ToconstmctplasmidvectorexpressionmitochondrialtargetedtruncatedformofAPE1(pcDNA—mtAPE1一HA)andtoinvestigatetheendothelialceUsurViValandprolif色rationafterperoxide—inducedoxidativestressinnuencedbytruncatedAPE1mitochodriaUyoVerexpression．3．ToinvestigatethepresenceofMTSinAPE1anditslo

5、cationintheproteinsequenceofAPEl．MaterialsandMethods1．A1terationofAPE1cellulardistributionafteroxidatiVestress：SubceUulardistributionofAPE1wasexaminedbylaserconfocalmicroscopyat0—3hinIRtreatedHOScellorperoxidetreatedHUVEC．WesternblotwasalsousedtoanalysistheAPE1proteinlevelinmitochondria

6、lextractstoconfirmthemorphologicalobserVation．2．Constructionandbi0109icaleff色ctsofplasmidVectoroVerexpressingtruncatedAPE1specificallyinmitochondria：First，theNterminaltruncatedAPE1andMTSwasamplifiedbyPCRandsplicedbyoVerlapextensionPCRtheninsertedintopcDNA3·1(+：betweenEcoRIandXhoIsites．T

7、hen，theplasmidVectorwassequencedandnamedaspcDNA—mtAPE1一HA．LaserconfocalmicroscopywasusedtoobserVethesubcellula】10calizationofrecombinantAPE1．WesternblotandAPEassaywere叩pliedtomeasureth(4第三军医大学硕士学位论文increaseofAPElproteinandenzymicactiVity1eVelinmitochondriaandnucleus．Subsequentl