HR24L基因过表达抑制细胞凋亡

HR24L1,2,1,2(1.,21,100083)[]HR24L;;[]:DNAHR24L:PCRHR24L,pDOR2neo,HeLa,;SouthernHR24LNorthernHR24LmRNA,,DNALadder,Western:HR24LHeLaHeLa,HeLa2HR24LHR24LmRNAHR24LBcl22,caspase23Bax,p53HR24L,:HR24L[]Q754[]A[]16712167X(2002)0120028205OverexpressionofHR24LgeneinhibitsapoptosisYUTing2Xi1,2,ZHUYing2Bao1,TONGTan2Jun2(1.MolecularBiologyLaboratory,PekingUniversityThirdHospital;2.DepartmentofBiochemistryandMolecularBiology,PekingUniversitySchoolofBasicMedicalSciences,Beijing100083,China)KEYWORDSHR24Lgene;Transfection;ApoptosisSUMMARYObjective:TostudytheeffectofhumanDNArepairgeneHR24Lonapoptosis.Methods:TheopenreadingframeofHR24LwasamplifiedbyPCRandwasclonedintotheretroviralvectorpDOR2neo.TransfectionofHeLacellswasperformedwiththerecombinantvector.Apoptosiswasin2ducedbyH2O2,sodiumbutyrateandserumstarvation.FACSandDNAagarosegelelectrophoresiswereusedtoobserveapoptosis.Westernblotanalysisexaminedapoptosis2relatedproteins.Results:HeLacellsweresuccessfullytransfectedwithHR24Lgene,andoverexpressionofHR24Lgeneresult2edinascendanceofBcl22,inhibitedtheexpressionofcaspase23andBaxandsuppressedtheapoptosiscausedbyH2O2,andserumstarvation,buthadnoeffectontheexpressionofp53andtheapoptosiscausedbysodiumbutyrate.Conclusion:OverexpressionofthehumanrepairgeneHR24Lcaninhibitapoptosis.[JPekingUniv(HealthSci),2002,34:28-32]DNA,DNADNA,DNA,DNA,,,HR24LRAD24RADI7DNA[1,2],:(30070232)SupportedbyNationalNaturalScienceFoundationofChina(30070232)82()JOURNALOFPEKINGUNIVERSITY(HEALTHSCIENCES)Vol.34No.1Feb.2002©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,RAD24[3]HR24LG2rad24L,[4]HR24L,HR24L,HR24LHeLa,HR24L11.1HeLa,HR24LpACT24L,BiolabpGEM2TPCRPromegaE.coliTopl0InvitrogenPEG8000FlukaSigmapDOR2neo,pBR322Rous(longtermi2nalrepeat,LTR),G418,Dr.MorgesternLandG418DulbeccosmodifiedEaglesmedium(DMEM)LipofectamineGib2coSantaCruzSu2persignalWestPicoWesternblotPierce1.21.2.1PCRHR24LHR24L,NetPrimer,:,52AATATGAATTCGACGAAATGAATCAGGTAACAGACT23;,52AATAGGAATTCTCTATGTCCCATCACTCTCGTAGT23pACT24LHR24L,:945min,941min,502min,722min,5,940.5min,561min,721min,25,7210min1.2.2HR24LpGEM2T500lPCR50l3molL-1NaAC(pH5.2),1ml,,-2030min,415min,75%(),100l,10gL-14,2.0kbHR24L,pGEM2T,EcoREcoRXba,pGEM2T2HR24L1.2.3HR24LpDOR2neopGEM2T/HR24LEcoR,HR24LcDNAEcoRpDOR2neo,HR24LcDNApDOR2neo,EcoR,Xba,Hind,pDOR2HR24L13%()PEG80001.2.4HeLa10%()FBS()100mgL-1100mgL-1DMEM375%()CO295%()63105/,50%60%,20lLipofectamine3g100l,,,30min,800l,PBS2,Lipofec2tamine18h,20%()FBS1m1,12h10%()FBS,1dG418800mgL-1,3,3,G4181.2mgml-1,,G418200mgL-11.2.561105,HeLa2HR24LHeLa2neoHeLa3,3,24h,61.2.6SouthernNorthernDNA,[5]RNATrizol1.2.7HeLa2HR24LHeLa2neoHeLa6,0.8mmolL-1,50mmolL-1,,,72h120h,PBS2,470%(),PBS2,0.5mlPBS,RNaseA100mgL-1,3730min,,DNACellFITCellCycleAnalysisVer292,HR24L©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.sion2.011.2.8DNADNA[5]10g,16gL-1,4Vcm-11.2.9Western[5],Western100g12%()SDS2PAGE,,p53Bcl22Baxcaspase23p53Bcl22Baxcaspase2322.1HR24LPCRPCR,pGEM2TpGEM2T2HR24LEcoREcoRXba,0.440.892.0kb,2.2HR24LEcoRpGEM2T2HR24L,HR24LcDNA,pDOR2neopDOR2HR24LEcoR2.01kbHR24LcDNAXba0.7kb,Hind1.9kb2.3pDOR2HR24LpDOR2neoHeLa,HeLa2HR24LHeLa2neoHeLa,HeLa2HR24L,HeLa2neo(1)1HeLa2hR24Ls,HeLa2neoHeLa(n=3,…xs)Table1CellcountingofHeLa2hR24Ls,HeLa2neoandHeLacells(n=3,…xs)104Cellst/d0123456HeLa1.001.930.213.930.335.980.418.240.6612.901.0816.601.23HeLa2neo1.001.900.193.910.316.010.428.400.6813.601.1716.401.33HeLa2HR24L1.001.6080.203.610.295.520.397.800.6311.500.98314.701.2533P0.05,comparedwithHeLa2neoandHeLacells,ttest12.4Southern3DNA,EcoRSouthern,,39.0kbHR24L,HeLa2HR24L2.0kbHR24L,HeLaHeLa2neo(1)HR24LcDNAHeLaa,HeLa2hR24L;b,HeLa2neo;c,HeLa11SouthernhR24LcDNAFigure1SouthernblotanalysisofhR24LcDNAintegration2.5HR24LmRNAHeLa,HeLa2HR24LHR24LmRNAHeLa2neoHeLa(2)HR24LHR24LmRNA,HR24LmRNAa,HeLa;b,HeLa2neo;c,HeLa2hR24L12NorthernHeLaHeLa2neoHeLa2hR24LhR24LFigure2NorthernblotanalysisofHR24LexpressioninHeLa,HeLa2neo,HeLa2hR24L03()JOURNALOFPEKINGUNIVERSITY(HEALTHSCIENCES)Vol.34No.1Feb.2002©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.2.6(AP),APHeLa2HR24LsAPHeLaHeLa2neo(P0.05,2),HR24L2HeLa,HeLa2neoHeLa2HR24Ls(n=4,…xs)Table2FlowcytometricanalysisofapoptosisinducedbydifferentagentsinHeLa,HeLa2neoandHeLa2HR24Lscells(n=4,…xs)TreatmentPercentareaofapoptoticpeakHeLaHeLa2neoHeLa2HR24LsH2O246.84.745.34.423.62.83Sodiumbutyrate43.64.543.64.144.65.1Serumstarvation46.84.748.34.923.43.133P0.05,comparedwithHeLabythesametreatment,ttest12.7DNA72h120hDNA,,HR24L,(3)a,HeLa;b,HeLa2neo;c,HeLa2hR24L;M,DNA/Hind3DNAFigure3GelelectrophoresisofDNAfragmentationinducedbydifferentcauses2.8WesternWestern,p53pDOR2neopDOR2HR24LHeLaBcl22pDOR2HR24LpDOR2neoHeLaBax,pDOR2HR24LpDOR2neoHeLa,caspase23pDOR2HR24LspDOR2neoHeLa(4)a,HeLa;b,HeLa2neo;c,HeLa2hR24L.4WesternblotFigure4WesternblotanalysisoftheexpressionofdifferentapoptosisrelatedproteininHela,Hela2neo,Hela2hR24Lcells3pACT24L,PCRHR24L,HR24LpDOR2HR24L,HR24LcDNA,HR24LHeLaHR24LmRNA,,,HR24LHR24L,HR24L,HR24LBcl22,[6,7],Bax,Bcl22,,Bcl22,Bcl22(Bcl2XL)/BaxBcl22(Bcl213,HR24L©1995-2004TsinghuaTongfangOpticalDiscCo.,