机体无时无刻不受到内外环境条件变化的应

ISSN100727626CN1123870PQChineseJournalofBiochemistryandMolecularBiology2002418(2):2342391),1)3,2),2),2),2)(1),300050;2),100850),.123256,8313%110%.11NE(P0105),6(MrPpI:4917kDP718,3813kDP519,3711kDP616,2913kDP714,1817kDP611,1815kDP717),4(MrPpI:4716kDP515,3119kDP414,2616kDP416,3312kDP811),1(MrPpI:1914kDP619)..,,,Q334,Q71Two2dimensionalPolyacrylamideGelElectrophoresisAnalysisofStressedCardiomyocytesProteomeDUANHai2feng1),QIANLing2jia1)3,YINGWan2tao2),QIANXiao2hong2),YANGHe2yi2),LUYing2)(1)InstituteofHygieneandEnvironmentalMedicine,AcademyofMilitaryMedicalSciences,Tianjin300050,China;2)BeijingInstituteofRadiationMedicine,Beijing100850,China)AbstractHighresolutiontwo2dimensionalpolyacrylamidegelelectrophoresis(22DE),followedbycomputer2assistedimageanalysiswasusedtoscreenproteinpatternsofnormalandnorepineprine(NE)2stressedcardio2myocytesforquantitativeandqualitativedifferencesinproteinexpression.123256proteinspotswerere2solvedinnormalcardiomyocytesandamatchrate8313%110%wasachieved.Theresultsalsoshowthat11proteinspotsdisplayedquantitativechangesinexpressionafterinductionofNE(P0105),ofwhich,6(MrPpI:4917kDP718,3813kDP519,3711kDP616,2913kDP714,1817kDP611,1815kDP717)decreasedinabundanceand4(MrPpI:4716kDP515,3119kDP414,2616kDP416,3312kDP811)showedhigherexpres2sionand1(MrPpI:1914kDP619)onlyexpressedinNE2inducedcells.Theseproteinsmaybeinvolvedinthecardiovascularresponsetostressandthestress2inducedcardiovascularinjury.Keywordscardiomyocyte,proteome,two2dimensionalpolyacrylamidegelelectrophoresis,stress:2001206204,:2001208216(No.G20000569)3Tel:[phone],E2mail:[e-mail],,197311,Received:June4,2001;Accepted:August16,2001SupportedbySpecialFundsforMajorStateBasicResearchProgramofChina,No.G200005693CorrespondingauthorTel:[phone]E2mail:[e-mail].,.,,[1].,--(HPA)(SNS).(NE),,.,[2,3].,NE..,,.,,.(proteome)MarcWilkins[4]1995,.,.,,,[5-7].,(22DE),22DE.NE,.1111;;MEMGIBCOBRL;254.30%(acrylamide)P(291,313%C)Bio2Rad;BrdU(TEMED)TritonX2100CHAPSE264leupeptinbenzamidineHClpepstatinSigma;Pharmalyte3210(ultraurea)IPG(pH=310,L=18cm)SDSAmershampharmacia;DTTPromega;PMSFMerck;TrisDNaseRNase;(iodoacetamide)Fluka;.11213dWistar(),IPGphor(AmershampharmaciaBiotech),Protein(Bio2Rad),(Sigma),(Sigma),NAPCOCO2(Precision).PDQUEST610(Bio2Rad).113Simpson[8].13dWistar,,,20%011mmolPLBrdUMEM.100P.4d,,,24h,.11422DE,90%,,PBS3,5mmolPLEDTA15min.EDTA,50ml,,,Tris2HCl(10mmolPLTris2HClpH715,0136mgPmlE264,20molPLPMSF,0134mgPmlpepsta2tin,516mgPmlbenzamidineHCl1mgPmlleupeptin),5,DNaseRNase,10min.30min,,(89molPL,4%CHAPS,40mmolPLTris65mmolPLDTT).G250[9].4,12000rPmin20min.115pHSanchez[10].(810molPL,2%CHAPS,218%DTT,015%IPG,),350l(IPG18cm,310L).IPG,IPG(),.IPG,,IPGphor(AmershampharmaciaBiotech),20,45kVh48kVh.,IPG(115molPLTris,6molPL,30%,2%SDS,)32ml,15min2.,DTT100mg,0145g.116SDS2PAGE5322:IPGconversionKit,200mm200mm1mm,13%.IPG,015%.Tris2Gly2SDS,1415,30mA,.[11],20.117GS2710,1200ppi(pixelperinch),8417mPpixel.PDQUEST610.,,(),,.(2DCalibration).MicrosoftExcel97,Students2Test.221110(100ml,38cm2),107,50l.651920122gPl,107292g.21222DE18cmIPG,123256.Fig11.Fig.122DPAGEofnormalcardiomyocyteelectrophoresisofcelllysates(110gprotein)wasperformedusingIPG2DaltequipmentandmethodsIEFwascarriedoutinpH=3-10Lstripsasthefirstdimension,andtheseconddimen2sionalverticalSDS2PAGEconcentrationwas13%constant.Thegelwassliver2stainedandscannedbythedensitometerGS271021322DE,,8313%110%.Fig122,a.Fig13ab.2143,22DEFig14.131962,132274.,48,18195kD.63218Fig.222DPAGEofnormalcardiomyocyteelectrophoresisofcelllysates(110gprotein)wasperformedusingIPG2DaltequipmentandmethodsIEFwascarriedoutinpH=3-10Lstripsasthefirstdimension,andtheseconddimensionalverticalSDS2PAGEconcentrationwas13%con2stant.Thegelwassliver2stainedandscannedbythedensitometerGS2710Fig.3MatchmapofFig.2aandbTheredandthebluecirclesstandfortheproteinspotsinFig.2aandFig.2brespectively.Thespotsthatwerelinkedwiththelinesshowthattheyhavebeenmatched.Thematchrateis8313%110%215PDQUEST610(Query),6,4,1.Fig15.Table1.Table1ProteinalterationsinducedbyNESpotpIMrPkDRelativespotintensity(%)NormalcellsStresscellsValueofP17.4029.3310.2890.0190.0640.0260.001328.1233.2160.1300.0330.2760.0090.000936.0718.7140.5510.0190.2060.0060.001746.8719.4280.1420.01657.7218.4830.9270.0060.1010.0070.000464.4331.9860.1430.0560.5010.0220.002974.6426.6000.0490.0240.1840.0530.020585.9738.3330.9290.0230.2100.0100.008496.5837.0630.3080.0130.0090.0020.0235106.1435.6030.0120.0100.1970.0280.0101117.8349.7190.3440.0430.0330.0120.00713AmershampharmaciaBiotechIPG2phor,,8000V,.,IPGphor,,,,;,7322:Fig.4Thesliver2stained22DEproteinmapsofcontrolandNE2inducedcardiomyocytesThearrowsshowthesignificantdifferenceinabundanceofspotsincontrolandNE2inducedcardiomyocytes.ThecircleshowsthespecificallyexpressedproteinspotinducedbyNEFig.5ProteinalterationsinducedbyNEThefiguresarethemagnifiedregionsofsliver2stained22DEproteinpatternsofcontrolandNE2inducedcardiomyocytes.ThearrowsshowthesignificantdifferenceinabundanceofspotsincontrolandNE2stressedcardiomyocytes.Thecircleshowsthespecificallye