中国生物化学与分子生物学报

ISSN100727626CN1123870PQChineseJournalofBiochemistryandMolecularBiology2004820(4):5455501,2),2)3,2),2),2),3),1),1)(1),100083;2),100730;3),100730)IMR290,,IMR290,.IMR290581355104712PDL().IMR290,,IMR290SDNA.IMR290.,IMR290,,.,,IMR290Q28ConstructionoftheModelofCaloricRestrictionRetardingReplicativeSenescencewithHumanDiploidFibroblastIMR290JINJun2hua1,2),ZHANGTie2mei2)3,ZHANGEn2yi2),HANCai2he2),WUXiao2meng2),XUXiao2dong3),ZHANGZong2yu1),TONGTan2jun1)(1)DepartmentofBiochemstryandMolecularBiology,PekingUniversityHealthScienceCenter,Beijing100083,China;2)BeijingInstituteofGeriatrics,3)Departmentofhaematology,BeijingHospital,MinistryofPublicHealth,Beijing100730,China)AbstractInordertoconstructthemodelofcaloricrestriction(CR)withhumandiploidfibroblastIMR290,IMR290wasculturedinnormalconcentrationglucose(510mmolPL,NG),lowerconcentrationglucose(310mmolPL,LG)andhighconcentrationglucose(2215mmolPL,HG)media,respectively.Biomarkersofreplicativesenescencewereassessinthismodel.TheaveragesoflifespanofIMR290underNG,LGandHGcultureconditionswere5510,5813and4712populationdoublinglevels(PDL),respectively.AlthoughthecellgrowthrateofIMR290atearlystageinLGculturebecamesignificantlyslowerthanthatinNGandHGculture,itsproliferativecapacitytothestimulationofgrowthfactor(EGF)wasalmostthesameascellsculturedinNGandHGmedia.Atlatestage,thepercentageofSphaseofcellcycleandthelevelsofunscheduledDNAsynthesesinLGgroupweresignificantlyhigherthanthoseinNGandHGgroups.Thepositiverateofsenescenceassociated2galactosidase(SA22gal)stainingatlatestageofLGgroupwassignificantlylowerthanthatcellsoflatestageinNGandHGcultrue.TheseresultssuggestedthatthemodelofcaloricrestrictioninvitrowasconstructedbyusingIMR290culturedinlowerglucoseconcentrationmediumandcaloricrestrictioncouldretardthereplicativesenescenceofIMR290.Keywordscaloricrestrction,cellularreplicativesenescence,humandiploidfibroblastIMR290:2004205210,:20042062103Tel:0102651322662676;E2mail:tmzhang537@hotmail:com,,19651,,Received:May10,2004;Accepted:June10,20043CorrespondingauthorTel:0102651226622676;E2mail:tmzhang537@hotmail:com1935McCay(lifespan)[1],[2],,30%50%,,©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.30%50%.,,.(caloricrestriction,CR).CR,,,,,.CR[3],CR,,CR.,..(populationdoublinglevel,PDL),[4],CR.,[5].[6]..CR,CR,.11.1IMR290(24PDL)ATCC,Nichols[7](1977).(FBS)HyClone.DMEM(EGF)GIBCO.(MTT)(DMSO)(hydroxyurea,HU)Sigma.ELISA2BrduBoehringer.Bio2Rad.Becton2Dickinson.2(senescenceassociated2galactosidase,SA22gal)(1mg/mlX2gal(Amresco),5mmolPLK4Fe(CN)6,5mmolPLK3Fe(CN)6,2mmolPLMgCl2,40mmolPLsodiumcitrate,pH610).1.2IMR290ATCCIMR29024PDL,[7],DMEM(510mmolPL,10%FBS,2mmolPL,l%,100UPml,100UPml),37,5%CO2.23d1,80%90%,0125%+013%EDTA,2104Pcm2.ATCCn=3132(logN2-logN1)+XPDL.nPDL,N2,N1,XPDL.IMR290(510mmolPL,NG)56PDL,Nichols[7].1.3,40%[2];(2%015%)[5];2215mmolPL[6].IMR29026PDL,2104Pcm2,DMEM(510mmolPL,NG),(310mmolPL,LG)DMEM(40%),(2215mmol/L,HG)DMEM,,.,,PDL50%,90%,50%90%[7].26PDL.,,10PDL,363836403637PDL,90%,505453584448PDL.1.4,1105P6,3,12h64520©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,120h,.2d1,120h.,,,5d95%,90%.,120h.1.5EGF,3103P96,8,,1%FBS72h,1%FBS200mgPmlEGF.72h,1%FBS+200ngPmlEGF.EGF16hMTT(50gPml),374h,PBS1,DMSO150lP,,A490.11670%80%,,75%,RNA(20gPml)3730min,(PI,50gPml),.117DNA,104P96,8,,1,015%FBS72h,.5mmol/LHU1,371h;200molPLH2O25mmolPLHU,371h;5%FBS5mmol/LHU,Brdu,3716h,ELISA2Brdu,A450PA650.1182(SA22gal),2105P6,3,,PBS2,3%15min,PBS3,SA22gal37(CO2)16h.(100)().119xs,t.22.1IMR290IMR290(Table1).IMR29055100175813018(,P0101)4712018PDL(,P0101)IMR290611%,2316%;IMR2901412%.Table1TheeffectofvariousconcentrationsofglucoseonlifespanofIMR290GroupsPopulationdoublinglevels(PDL)PercentageofincreasePdecreaseonlifespan(vsNG)Percentageofincreaseonlifespan(vsHG)NG(n=5)(510mmolPLglucose)5510017LG(n=6)(310mmolPLglucose)581301833,##+611+2316HG(n=5)(2215mmolPLglucose)471201833-141233P0101(vsNG);##P0101(vsHG)(Fig11),,,.23d1,34d1.50PDL,,,,,,1,.(50PDL),,56d1,5PDL.45PDL,5PDL.(Fig12),..160h72h48h.120h.2.2EGF7454:©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.Fig.1MorphologyandSA22galstainingforIMR290(magnification100)(A)CellsofearlystageinLGmediumandSA22galstainingnegative;(B)CellsofearlystageinNGmediumandSA22galstainingnegative;(C)CellsofearlystageinHGmediumandSA22galstainingnegativeexceptmarkedpositive;(D)CellsoflatestageinLGmediumandSA22galstainingpositive;(E)CellsoflatestageinNGmediumandSA22galstainingpositive;(F)CellsoflatestageinHGmediumandSA22galstainingpositiveFig.2GrowthcurveofIMR290CellsofearlystageinLGmedium;CellsofearlystageinNGmedium;CellsofearlystageinHGmedium;CellsoflatestageinLGmedium;XCellsoflatestageinNGmedium;CellsoflatestageinHGmediumEGF(Fig13).EGF,EGF75%51%110%.EGFFig.3TheeffectofEGFonIMR290cellsproliferationWithoutEGFstimulation;WithEGFstuimulation;CellsofearlystageinLGmediumchangedtoHGmediumwithEGFstimulation.ELG:cellsofearlystageinLGmedium;ENG:cellsofearlystageinNGmedium;EHG:cellsofearlystageinHGmedium;LLG:cellsoflatestageinLGmedium;LNG:cellsoflatestageinNGmedium;LHG:cellsoflate