ProteomechangesduringBMSCdifferentiationintophotoreceptor-likecells 窑BasicResearch窑 Proteomechangesduringbonemesenchymalstem celldifferentiationintophotoreceptor-likecells

Foundationitems: NationalNaturalScienceFoundationofChina soon.Recently,manyscholarshaveinducedBMSCto (No.81070715); InnovativePlatformFoundation of Fujian expressaspecificmarkerinphotoreceptorcells.BMSC Province,China(No.2010Y2003) differentiationintophotoreceptorcellsiscomplexand FujianInstituteofOphthalmology,TheFirstAffiliatedHospitalof requiresaseriesofproteinchangesandinformationcontrol. FujianMedicalUniversity,Fuzhou350005,FujianProvince,China Currently,informationregardingthefeaturesofBMSCsand Guo-XingXu.FujianInstituteofOphthalmol- Correspondenceto: theunderlyingprotein control mechanismoftheir ogy,TheFirstAffiliatedHospitalofFujianMedicalUniversity, differentiationintophotoreceptorcellsisscarce.With Fuzhou350005,FujianProvince,[email protected] emergingtechnologyandthedevelopmentoftheproteome, Received:2011-05-27Accepted:2011-09-19 proteomicstechnologieshavebeenwidelyappliedtostudy oftheinductionofBMSCdifferentiation.Inthepresent Abstract study,BMSCsare inducedanddifferentiatedinto ·Humanbonemarrowstemcell(BMSC)maybedirectedto photoreceptor-likecellsoftheretinabysimulatingthe differentiateintomultiplecelltypes,includingadipocyte, microenvironmentoftheretina .Two-dimensional chondrocyte,osteocyteandphotoreceptor,amongothers.At differencegelelectrophoresis(2D-DIGE)technologyis present,littleisknownaboutthefeaturesoftheBMSCand adoptedtocomparetheproteinsexpressedduringthe theproteincontrolmechanismunderlyingtheirdifferentiation BMSCdifferentiationintophotoreceptor-likecellsto intophotoreceptor-likecells.Inthepresentstudy,BMSCsare establishadifferentialproteinexpressionprofile.Atotalof inducedtodifferentiateintophotoreceptor-likecellsinan 37proteinspotswithsignificantdifferentialexpressionare modelsimulatingthe microenvironment.Upto32 found.Upto32differentialproteinsarecompletely proteinsareidentifiedanddifferentiallyexpressedthrough two-dimensionaldifferencegelelectrophoresisand identifiedthroughmatrix-assistedlaserdesorption/ionization matrix-assistedlaserdesorption/ionizationtime-of-flightmass time-of-flightmassspectrometry(MALDI-TOF-MS) spectrometrytoestablishadifferentialproteindatabasefor analysis. photoreceptor-likecellsfromBMSC-induceddifferentiation. MATERIALSANDMETHODS Westernblotanalysisfurtherconfirmstheexpressionofsome HumanBMSCsstrainsculturedanddifferentiatedto oftheidentifiedproteins.Thepresentstudyproposesthe photoreceptor-like cell HumanBMSCstrainswere totalproteinexpressionandpossiblemolecularmechanism incubatedinmesenchymalstemcellmedium(MSCM)inan duringthedifferentiationofBMSCsintophotoreceptorcells. incubatorwith100%humiditycultured.Theliquidwas · KEYWORDS:bonemarrowstemcell;inducedto changedeverythreedays.Whentheculturesreached80% differentiate;photoreceptor-likecells 85%confluence,thesolutionwasdigestedwith0.25% DOI:10.3980/j.issn.2222-3959.2011.05.02 trypsinandsubculturedintheproportionof1:2. P4humanretinalpigmentepithelial(RPE)cellswere HongY,XuGX.Proteomechangesduringbonemesenchymalstem culturedin10%fetalbovineserum+RPMI1640culture celldifferentiationintophotoreceptor-likecells medium.WhentheRPEcellsreachedapproximately95% 2011;4(5):466-473 confluence,thecultivationliquidwascollectedandfiltered usinga0.22 mmeshandmixedwithMSCMcontaining20 滋 INTRODUCTION ng/mleachofbasicfibroblastgrowthfactor,epidermal umanbonemarrowstemcells(BMSCs)are growthfactor,andbrain-derivedneurotrophicfactorata H multipotentstemcells.Theycanbedirectionally ratioof1:4topreparetheconditionedmedium. differentiatedintofat,cartilage,bone,andmusclecells,and HumanBMSCstrainsfromP2toP4wereobtainedfor

466 陨灶贼允韵责澡贼澡葬造皂燥造熏灾燥造援 4熏晕燥援 5熏 Oct.18, 圆园11 www.IJO.cn 栽藻造押8629原愿圆圆源缘员苑圆 8629-83085628 耘皂葬蚤造押陨允韵援圆园园园岳员远猿援糟燥皂 experimentation.TheculturedBMSCsintheconditioned respectively,andplacedintotwoexpandedEppendorftubes mediumwereusedasinductedgroup,whereastheMSCM and25 geachofthesamplesweremixedinonetube. 滋 culturewasusedasthenegativecontrol.Aninvertedphase Then,Cy3andCy5dyeswereaddedtoeachtube, contrastmicroscope wasusedtoobservecellular respectively,andCy2dyewasaddedintothemixedsample. morphologyanddifferentiationinbothgroups. Lysine(1 L)wasaddedwhenicereactionwithoutany 滋 Immunocytochemistry Thecelladhesionsheetswere radiationwasmade,andtoterminateicereaction.After collectedat3,5,and7daysafterinduction,fixed,andthen mixingtheloadingsolutionsevenly,theywerecentrifuged lysedwith1%Triton-100.Primaryanti-rhodopsinantibodies for8minutesat12000 g,andtheupperlayerwasobtained 伊 wereaddedataratioof1:200.Thesheetsweremaintained forisoelectricfocusing.From300Vfor45minutes,700V overnightat4℃.Thesheetswerewashedwithphosphate- for45minutes,1500Vfor1.5hours,andthenincreasedto bufferedsaline(PBS).Biotinylated,horseradishperoxidase 9000V,toreachatotalof9000Vh.Then,theimmobilized (HRP)-conjugatedanti-immunoglobulinsecondaryantibodies pHgradientstripequilibrationwas performed. The wereaddedtotheculturefor30minutesat37℃ ,andthen conditionsforthesodiumdodecylsulfatepolyacrylamidegel theywerewashedwithPBS.Thesampleswerestainedwith electrophoresis(SDS-PAGE)wereasfollows:S1)2-2.5 3,3'-diaminobenzidineandhematoxylinatroomtemperature W/gelfor45minutes,S2)17W/gelforaround4.5hours, forcolordevelopment.Foreachindex,5microscopefields andS3)bromophenolbluewasatthebottomat10 ℃ wererandomlychosentocalculatethepercentageof withoutanyradiation.Forstaining,theproteinswere positivelystaining. separatedbasedonthevariationsinisoelectricpointand Real-timePCR Primerexpress2.0softwarewasadopted molecularweight(MW).The2-DEgelwasstainedwith todesigntheprimerandprobe.AnABI3900table-type Deep TM TotalProtein .Then,theimageswere high-fluxDNAsynthesizerwasusedtosynthesizethe sannedwithaTyphoon9400atmaximumgrayvalueof primer.Thecellswithmaximuminducedpositivestaining(7 60000-90000. days)wereadoptedastheexperimentalgroup.BMSCs TreatmentofProteinSpots Imagemaster7.0wasusedto culturedwithMSCMwereconsideredasthecontrolgroup. analyzethespotsthathadmorethan1.5-folddifferencesin Then,1 105 cellswerecollectedforeachgroup.Trizolwas intensity.Theselectedproteinspotsfromthegelwere 伊 usedtoextracttotalRNAfromthecells.ARevertAidTM placedinEppendorftubesfordehydrationafterCoomassie FirstStrandcDNASynthesisKitwasadoptedtoreverse- staininganddestaining.Then,thespotswereswelledin transcribethetotalRNAandsynthesizefirststrandcDNA 20ng/ Lofice-coldtrypsinfor30minutes,and10 Lof 滋 滋 usinganSYBR ® PremixExTaq-TM kit.AThermalCycler coveringliquidwasaddedat37℃ forenzymecleavagefor Dice ® Real-TimeSystemwasusedforreal-timePCR. 12-16hours.Thespotswereultrasonicatedfor15minutes. Predenaturationwasperformedat95 ℃ for30secondsand Then,thecoveringliquidwasaspirated,andanextraction at95 ℃ for5seconds.Annealingwasconductedfor40 solutionwasadded.Thespecimenswereshakenfor10 cyclesat60℃ for30seconds. minutestodehydrateandaspiratetheextractionsolution. 2D-DIGEandMALDI-TOF-MSanalysis Thesolutionwasthenvacuum-driedandkeptat-20℃.Then, ProteinExtraction Approximately1 107 cellswerecollected 1.5 Lofheavysolutionwasaddedintothedriedsampleto 伊 滋 fromtheexperimentalandcontrolgroups.Theprotein dissolvecompletelythepeptidesegments.Sampleswere extractionwasconductedasfollows:Thecultivationliquid repeatedlyaspiratedintothesmallholeofasteeltarget wasremovedandthecellswerewashedthricewithPBS. severaltimes.Thespecimenswerefedintoamass Thecellsweredisintegratedwithacelllysissolutionof spectrometerfordetectionafter0.5 Lofgroundsubstance 滋 ACKlysisbufferandultrasonicatedinanicebath.Thecells wasaddedintosamplesandwerecompletelydried. werefractionatedthroughcentrifugationat12000rpmfor20 ProteinDetermination AnABI4800MALDI-TOFMass minutesat4℃.Thesupernatewascollectedandprecipitated SpectrometerfromUSwasusedtoconductthemass overnightat-20 ℃ aftertheadditionofacetone.Then,the spectrographicanalyses.Theconditionswereasfollows:UV solutionwascentrifugedat12000rpmfor2minutesat4℃ lightat355nm,arepetitionrateof200Hz,anaccelerating andwasthenallowedtoprecipitate.Theproteincontentwas voltageof20KV,andanoptimumresolutionof1500Da. determinedusinga2D-Quantkitbasedonthemanufacturer's Thesignalswererecordedwithinamassscanningrangeof instructions. 700to3200Da.Theautonomouspeakoftrypsinogenwas 2D-DIGEtwo-dimensionalelectrophoresis Todetermine usedastheinternalstandardtonormalizethemass theintensityofDIGEfluorescenceat4℃,50 gofproteins spectrometer.Allmassspectrogramswereobtainedin 滋 wereobtainedfromtheexperimentalandcontrolgroups, defaultmode. 467 ProteomechangesduringBMSCdifferentiationintophotoreceptor-likecells TheMascotdistillersoftwarewasusedtofilterthebaseline peakandtoidentifythesignalpeak.TheMascotsoftware fromMatrixSciencewasusedtosearchtheIPIdatabaseand toidentifyrelatedproteinsandtheirfunctions.Thesearch conditionswereasfollows:thepeptidemasswaslimitedto within800-4000DaandthemaximumallowableMWerror ofpeptide fragmentwascontrolledin 50ppm.The 依 enzymaticfragmentwasselectedincompletelyasone,and theerrorrangesoftheapparentPIandMrwereunlimited. Thespecieswashuman,andtheMHandmonoisotopicwere selectedasions.Thefixedmodificationwassetto carbamidomethylwhereasthevariablemodificationwasset Figure1P3BMSCsshowedclusterandfishblock-like tooxidation. morphology,andgrowthofclonyformingunit-fibroblastic7d Westernblotanalysis Proteinconcentrationwasadjusted afterpassage basedonthetotalproteinconcentrationsoftheexperimental and controlgroups.Equivalentproteinsampleswere obtainedandseparatedbySDS-PAGE.Thespecimenswere transferredontopolyvinylidenedifluoridemembranesand blockedwith5%skimmilkpowder.Rabbitanti-human zyxinantibodiesandrabbitanti-human -tropomyosin 茁 antibodieswereadded.Then,HRP-labeledsheepanti-mouse secondaryantibodies(ZhongshanGoldenbridgeBiotechnology Co.,Ltd.)wereadded.Mouseanti-human -actinmonoclonal 茁 antibodieswereusedastheinternalcontrol,followingthe2 Figure2 BMSCsdisplayedbipolarormultipolarinthe proceduresusedabove.QuantityOnewasusedtoconducta morphology7dafterinduction渊伊200冤 semiquantitativeanalysisofthereactionbankandrecordthe measuredgrayvalues. RESULTS MorphologicChangesinBMSCsduringDifferentiation intoPhotoreceptor-likeCells BMSCsfromP2toP8had uniformlyfusiformconfigurationsandgrewrapidly.After subculturingfor2days,thenumberofcellsincreased logarithmically.After4-5days,thecellsgrewasclone-like clusterorfishstockwithupto80%-90%confluence.Inthe controlgroup,aftertheBMSCswereinducedfor2days,the cellularmorphologygraduallychanged.Theircellsshrank, Figure3ICHstainingdetectedtheexpressionofrhodopsin andneuritesformed.Thecellsevidentlypartiallyshrank 5dafterinduction after5days,andthecellularappendagespresentedtriangular orsimplebipolarandmultipolarcells.After7days,some Inductionratesofthepositivecellsat3,5,and7dayswere cellswerefusiform,andthebipolarandmultipolarcells 4.83% 0.29%,25.36% 0.32%,and28.87% 0.43%, becameevidentlylonger(Figure1,2).Synapses,similarto 依 依 依 respectively(Figure3,4).Inthecontrolgroup, thoseinconeandrodcells,werefoundintheprotruding expressionwasnegative(Figure5).Therhodopsinand end.For1to3days,thecellpropagationratewasslower thanthatofthecontrolgroup,whichevidentlybeganslowly recoverinmRNAwereclearlyexpressedintheexperimental after4-5days.After7days,somecellsstartedundergoing group,whereasnoexpressionwasobservedinthecontrol apoptosis.TherewasnoevidentchangeintheBMSC group(Figure6,7).Thisshowsthatphotoreceptor-likecells configurationinthecontrolgroup. fromBMSCdifferentiationexpressthedistinctivemarkerof Immunohistochemistryofinducedcellsandreal-time normalphotoreceptorcells,andtheprocesscanbeusedas PCRidentification Immunohistochemistrywasusedto modelforculturinghumanphotoreceptorcells visualizerhodopsinexpressionintheexperimentalgroup. (Table1). 468 陨灶贼允韵责澡贼澡葬造皂燥造熏灾燥造援 4熏晕燥援 5熏 Oct.18, 圆园11 www.IJO.cn 栽藻造押8629原愿圆圆源缘员苑圆 8629-83085628 耘皂葬蚤造押陨允韵援圆园园园岳员远猿援糟燥皂 Table 1 Primers for real-time RT-PCR name Primer sequence（forward and reverse） Product size Rhodopsin 5'-CGAGCGGTACGTGGTGGTGT-3' 445bp 5'-GGAGCCCTGGTGGGTGAAGA-3' Recoverin 5'-TGTGTTCCGCAGCTTCGATT-3' 369bp 5'-TGAGGCTCAAACTGGATCAG-3' β-actin 5'-AAAGACCTGTACGCCAACACAG-3' 556bp 5'-TTTTAGGATGGCAAGGGACTTC-3'

Identificationofproteinby2D-DIGEdimensional electrophoresis Toensuretherepeatabilityofthe 2D-DIGE,theexperimentswereperformedintriplicate.The resultsindicatethattheexperimentishighlyrepeatable.A totalof1914isoelectricpointswereobtainedin4-7proteins afterthe2D-DIGEgelwasscannedatthreedistinct wavelengths.Basedonthestatisticalanalysis,therewere37 proteinspotswithmorethan1.5-folddifferences,and32 differential wereidentifiedthrough mass spectrometry.Amongthem,11proteinswereupregulated Figure4ICHstainingdetectedtheexpressionofrhodopsin and21proteinswere downregulatedduringBMSC 7dafterinduction differentiation(Figure8).Table2lists therelated informationaboutthe32proteinspotsandtheMWgiofthe proteinspotsfromlefttoright. Identificationoffunctionalclassificationofprotein The 32differentiallyexpressedproteinswereclassifiedinto7 categories:cytoskeletalproteins,molecularchaperones, kinasesforenergymetabolism,signaltransductionpathway- relatedproteins,cellproliferationproteins,differentiation andapoptosis-relatedproteins,calcium-bindingandion channelproteins,andotherproteins. VerificationofWesternblotanalysisresults Inthe Figure5 ICHstainingfortheexpressionofrhodopsinin presentstudy,Westernblotanalysiswasperformedtoverify controlBMSCs furtherthedifferentialexpressionofsomeproteins.Positive bandsat82and33kDaintheexperimentalgroupwere detected.Thesevalueswereslowerthanthatofthecontrol group,indicatingthatzxyinand -tropomyosinwere 茁 downregulatedafterinduction(Figure9). DISCUSSION Photoreceptorcells,namelyconesandrods,consistoffive parts,anoutersegment,connectingcilium,innersegment, Figure6Amplificationcurvesforexpressionofrhodopsinin body,andsynapses.Eachoutersegmentcoversmorethan theinductedandnon-inductedBMSCs 700flatmembranousdiscs.Theoutersegmentsofrodcells arecylindricalwhereastheyarecone-shapedinconecells. Theconfigurationsofsomecellsintheexperimentalgroup werealteredafterinductionfor2days.After5days,some cells shrankwithobviousproptosis.Therewere experimentalinsomecellsafter7days,andbipolarand multipolarcellsbecameevidentlylonger.Thesynapseis Figure7Amplificationcurvesforexpressionofrecoverinin foundattheprotrudingend,similartothoseinconeandrod theinductedandnon-inductedBMSCs cells.ImmunohistochemistryandRT-PCRverifiedthatthe 469 ProteomechangesduringBMSCdifferentiationintophotoreceptor-likecells

Figure8MSidentificationof32differentiallyexpressedproteinspots 渊NumbersrepresenttheSpotIDofidentifiedproteins袁Red fordown-regulatedandyellowforup-regulated冤

Figure9ExpressionofZxyinand 茁-tropomyosinbyWesternblot inducedcellsexpressedmarkersspecifictophotoreceptor evident,andtheupregulationofmyosin,mitochondrialheat cells,e.g.,rhodopsinandrecoverin;italsoshowedthe shock60kDaprotein1variant1,thioredoxindomain- differentiationoftheinducedcellsintophotoreceptorcells. containingprotein5isoform3,CytochromeB-C1 complex BMSCdifferentiationintophotoreceptor-likecellsisa subunit1,mitochondrial,andEF-handdomain-containing complexprocessthatrequiresaseriesofproteinchanges proteinD2wasobserved.Theseresultsindicateimportant andinformationadjustment.Proteomicstechnologycanbe adjustmentsduringtheinduction. usedtoprobethedifferentialexpressionofproteinsandthe Thep38/mitogen-activatedproteinkinase(MAPK)pathway mechanismofBMSCdifferentiationintophotoreceptor-like isanimportantandhighlyconservedsignalsystemforthe cellsinthedynamicstateandfromanintegralaspect.We externalsignalingofeukaryoticcells.Thispathwaybelongs establishedaninvitroinductionsystemofhumanBMSC tothesilk/serine-threonineproteinkinasefamily,whichis differentiationtophotoreceptor-likecells.Upto37protein presentinmostcells.MembersoftheMAPKfamilyconsist spotsthatareclearlydifferentiallyexpressedwerefound ofextracellularsignal-regulatedproteinkinase(ERK), using2D-DIGE.Additionally,32differentiallyexpressed p38MAPK,C-junaminoterminal/stressactivatedprotein proteinswereidentifiedbyMALDI-TOF-MSanalysis. (JNK/SAPK),andenzymesinthemitogen-activated Amongtheseproteins,11proteinswereupregulated, proteinkinases1(BMKl/ERK5)pathway [49].Thep38 whereas21proteinsweredownregulated.Adifferentially signalingpathway,activatedbyanexternalstimulus(suchas expressedproteinogramwasestablishedafter7daysof ultravioletradiation,radioactiveray,heat shockand BMSCdifferentiationintophotoreceptor-likecells.Inthe proinflammatoryfactorandphysiologicalstress),iscalled proteinogram,thedownregulationofp38,chainC,and theMAPKemergencesignalchannel.Thispathwayisone humanproliferatingcellnuclearantigen(PCNA)was oftherecentresearchhotspotsinsignaltransduction.

470 陨灶贼允韵责澡贼澡葬造皂燥造熏灾燥造援 4熏晕燥援 5熏 Oct.18, 圆园11 www.IJO.cn 栽藻造押8629原愿圆圆源缘员苑圆 8629-83085628 耘皂葬蚤造押陨允韵援圆园园园岳员远猿援糟燥皂

Table 2 The information on the 32 proteins identified by MALDI-TOF-MS MASCOT Algorithm Sequence Coverage Accession Spot name MASCOT Peptides MW PI NO ID P (%) （%） score 227 zyxin 138 3.7e-09 5 1.10E+01 62436 6.22 4508047 351 stress-induced-phosphoprotein 1 182 1.5e-13 6 9% 63227 6.4 5803181 366 NAG22 protein 168 3.7e-12 5 17% 56486 5.73 13186201 367 NAG22 protein 339 2.9e-29 5 14% 56486 5.73 13186201 369 NAG22 protein 415 7.4e-37 6 15% 56486 5.73 13186201 376 NAG22 protein 196 5.8e-15 4 14% 56486 5.73 13186201 379 prelamin-A/C isoform 1 precursor 257 4.6e-21 18 32% 74380 6.57 27436946 381 zyxin 203 1.2e-15 4 9% 62436 6.22 4508047 395 unnamed protein product 133 4.8e-23 3 10% 53642 5.08 10438296 400 PREDICTED: hypothetical protein isoform 4 284 9.3e-24 9 22% 61784 4.93 114581740 403 unnamed protein product 314 9.3e-27 20 32% 65037 5.47 221039676 675 T-complex protein 1 subunit beta isoform 2 316 5.8e-27 5 15% 53027 6 311771535 850 thioredoxin domain-containing protein 5 isoform 3 403 1.2e-35 7 22% 36725 5.32 224493972 945 GDP dissociation inhibitor 2, isoform CRA_a 616 5.8e-57 21 49% 48680 7.51 119606836 964 reticulocalbin-1 precursor 167 4.6e-12 6 22% 38866 4.86 4506455 986 Chain A, Bm-40, FsEC DOMAIN PAIR 313 1.2e-26 9 35% 27852 5.53 2624793 1032 reticulocalbin-1 precursor 268 3.7e-22 8 31% 38866 4.86 4506455 1070 PREDICTED: -like capping protein isoform 9 205 7.4e-16 7 21% 38779 5.88 55597035 1141 beta tropomyosin 391 1.8e-34 17 38% 29980 4.7 6573280 1146 tropomyosin alpha-4 chain isoform 1 405 7.4e-36 13 31% 32874 4.69 223555975 cytochrome b-c1 complex subunit1, mitochondrial 1025 341 1.8e-29 7 31% 53297 5.94 46593007 precursor 1248 Chain C, Human Pcna 159 2.9e-11 9 42% 29072 4.53 2914385 EF-hand domain-containing 1344 551 1.8e-50 13 38% 26795 5.15 20149675 protein D2 1383 cathepsin D preproprotein 267 4.6e-22 6 15% 45037 6.1 4503143 1393 cathepsin D preproprotein 214 9.3e-17 7 15% 45037 6.1 4503143 1395 glutathione S-transferase omega-1 isoform 1 181 1.8e-13 5 19% 27833 6.23 4758484 1491 cathepsin B 172 1.5e-12 3 30% 17428 5.44 741376 1513 keratin 1 127 4.6e-08 6 16% 66198 8.16 11935049 Chain A, Ternary Complex y Of An Crk Sh2 Domain, 1533 Crk-Derived Phophopeptide, And Abl Sh3 Domain B 81 0.0017 4 46% 12122 6.79 253722243 Nmr Spectroscopy 1641 mitochondrial heat shock 60kD protein 1 variant 1 201 1.8e-15 5 15% 60813 5.83 189502784 1687 myosin, light polypeptide 6, alkali, smooth muscle and 120 2.3e-07 4 25% 13264 4.47 119617308 non-muscle, isoform CRA_d 1888 alpha-2-macroglobulin 462 6.9e-040 6 5% 168953 5.71 157954061 1888 alpha-2-macroglobulin 166 5.5e-11 4 4% 168953 5.71 157954061 （2）

Throughthecascadereactionofupstreammulti-stage showstheevidentdownregulationofp38duringBMSC ,anextracellularsignalistransferredintracellularly, differentiation.Theresultshowsthatp38signalingpathway resultingintheactivationofp38.Thissignalpathway playsanimportantroleinregulatingthedifferentiationof controlstheactivityofmanydownstreamtranscription BMSCintophotoreceptorcells. factors,suchastranscriptionfactorsinvolvedinactivation, ChainCandhumanPCNA PCNAisaproteinthat NF- B,andgrowtharrest,aswellasDNAdamagegene. participatesinDNAreplicationduringcellproliferation. 资 Recentstudieshaveshownthatthep38phosphorylated Thisproteinislocalizedinthecellnucleusandisacyclic pathwayisanimportantcommonsignalpathwayforcell trimerthatconsistsofthreesimilarsubunits.PCNAisnot differentiation.Baifoundthatthep38pathwayplaysarole expressedincellsduringG0-G1.PCNAupregulationoccurs inpromotingMSCdifferentiationintoepidermoidcells.The duringlateG1andpeaksduringtheSperiod,whereasrapid blockageofupstreamsignalRhomayincreasetheactivation downregulationisobservedduringG2-M.Atthestartof ofthep38pathway,andpromotesMSCdifferentiationinto eukaryoticDNAreplication, PCNAenablesDNA epidermoidcellstoacertaindegree.Thepresentstudy polymerase andusestheDNAslideinitslooptoinstruct 啄 471 ProteomechangesduringBMSCdifferentiationintophotoreceptor-likecells thecontinuoussynthesisoftheguidingchain.PCNA,which increaseinthenumberofnecroticandapoptoticcellsamong isonlypresentinnormalproliferativeandtumorcells,plays theBMSCs,whichwilllowerthemultiplicationrateofcells. animportantroleininitiatingcellproliferationandisa Thioredoxindomain-containingprotein5isoform3(Trx) betterindexforreflectingthevegetativestateofcells.In Trxisahighlyconservativeandubiquitouslyexpressed addition,apoptosis-relatedproteinp21competesforthe smallproteinwithamolecularweight13kDa,andanactive bindingsiteofDNApolymerase ofPCNAtoinducethe disulphidecenterwasusedtocatalyzetheoxidation- 啄 cellstoexitG1andentertheSperiod,resultinginthedirect reductionreaction.Trxmaintainstheoxidationreduction blockageofDNAsynthesisandinducingcellapoptosis. equilibriumofcellsbyfacilitatingthescavengingoffree

Recently,therehasbeenincreasinginterestinPCNA radicals,enablingthecelltowithstandH2O2anditstoxic studies.Inthepresent experiment,evidentPCNA effectstoreduceapoptosis.Thisproteinformsacomplex downregulationwasobservedduringthedifferentiationof withASKl,therebyinhibitingASKIandpreventing BMSCsintophotoreceptor-likecells.BMSCshavestrong apoptosis [48].Additionally,Trxplaysanimportantrolein proliferativecapacities.BMSCsincreaselogarithmically2d signaltransductionbyregulatingMAPK,aswellasthe afterthepassageculture.After4-5d,theBMSCsproliferated activationoftranscriptionfactorandotherfunctions.Inthe byupto85%-90%,whereasthemultiplicationcapacityof presentexperiment,BMSCswereinducedtodifferentiate theinducedcellswasevidentlydecreased.Theseresults intophotoreceptor-likecells.Theoxidation-reductionsystem conformtothetrendofPCNexpression. ofcellswasactivatedbecauseofthechangesinmetabolism. Mitochondrialheatshock60kDaprotein1variant1 Trxwasevidentlyupregulated,whichindicatesthatitplays Heatshockproteins(HSPs),agroupofhighlyconserved aroleinmaintainingtheoxidation-reductionequilibriumof proteins,existwidelyinprokaryoticandeukaryoticcellsand cellsandthepreventionofapoptosis. theyarealsocalledstressproteinsormolecularchaperones. CytochromeBC1 complexsubunit1,mitochondrial The Approximately70%-80%HSP60ofeukaryoticcellare mitochondrialrespiratorychainconsistsoffourcomplexes, mainlylocatedinthemitochondria,and20%-30%isinthe includingcytochromeC,andcoenzymeQ.Thecytochrome [42] endochylema .Astwoofthemostimportantmolecular B-C1 complexreferstocomplexIII,alsoknownasthe chaperoneproteinsinthemitochondria,2HSP60heptamer CoQH2-cytochromeCreductasecomplex.Thiscomplex and1HSP10heptamerformahigh-molecular-weight transmits4H+totheintermembranespaceforoxidation- polymercalledlargerespirator,whichisanimportant reductionwhenapairofelectronsistransmitted.This structureformitochondrialproteinrefoldingandrestoration. complexmovescontinuouslyinthemitochondrialinner Liang .foundthatHSP60intheendochylema membrane,soithasnostablestructure.Thisproteinwas participatedinthesynthesisandprotectionofcytoskeletal evidentlyupregulated,whichindicatesthatitparticipatesin proteins,suchasmicrotubulinandactin.HSP60hasadual theoxidation-reductionreaction. regulatoryfunction,anti-apoptosisandpro-apoptosis.HSP60 EF-handdomain-containingproteinD2 TheEFh-domain [50] physiologicallyplaysaroleinanti-apoptosisbymaintaining containingproteinD2wasfirstfoundinlymphocytes and thenormalconformationandfunctionofanti-apoptotic laterindifferenttissues.Itishighlyexpressedinmarrow [51]. factors,therebyinhibitingtheactivationofpro-apoptotic TheB-cellreceptorisadjustedtoinduceimmatureB-cell factorsandreducingtheformationofreactiveoxygen andapoptosisofitsoriginalB-cell,andBCL2L1isadjusted speciesinthemitochondria.Inthepathologicstate,HSP60 tocontrolnaturalapoptosis.Zhang [52] foundthatthe expressionanditssubcellularlocalization,aswellasitspro- EF-handdomain-containingproteinD2isupregulatedduring apoptoticeffectsarechanged.Manystudieshaveindicated MSCdifferentiationintoosteoblastandprovedthatitplays thatHSP60inisastressproteinreleasedbyBMSCsthatare animportantroleduringBMSCdifferentiation.Inthe [44,45] closetoundergoingnecrosisandapoptosis .HSP60 presentexperiment,EFh-domaincontainingproteinD2 expressionthereforeindicatesthepresencesofnecroticand clearlyincreasedafterinduction,indicatingthatitplaysan apoptoticcellsamongtheBMSCs.HSP60protein1variant importantroleduringBMSCdifferentiation. 1wasevidentlyupregulatedinthemitochondriaduringthe Myosin,lightpolypeptide6,alkali,smoothmuscleand differentiationofBMSCsintophotoreceptor-likecells, non-muscle,isoformCRA_d Myosin,whichexistswidely illustratingthatthereweremorenecroticandapoptoticcells inmuscleandnon-musclecells,combinesandinteractswith amongtheinducedcellsthanthenon-inducedBMSCs.In actintohydrolyzeadenosine-5'-triphosphate,guanosine-5' thepresentexperiment,weobservedareductioninthe -triphosphate,cytidinetriphosphate,andectenzyme.This multiplicationrateoftheinducedcellscomparedwiththat proteinsuppliespowerformusclecontraction,cytoplasmic ofthenon-inducedBMSCs.Thismaybecausedbythe streaming,organellemovement,materialtransport,and 472 陨灶贼允韵责澡贼澡葬造皂燥造熏灾燥造援 4熏晕燥援 5熏 Oct.18, 圆园11 www.IJO.cn 栽藻造押8629原愿圆圆源缘员苑圆 8629-83085628 耘皂葬蚤造押陨允韵援圆园园园岳员远猿援糟燥皂 mitosis.Myosinisalsocalledthemolecularmotorof Furtherinvestigationshouldbeperformedtoascertainthe cytoskeleton.Thepresentstudyfoundthatmyoglobulin functionsoftheseproteins. significantlyincreasedduringthedifferentiationofBMSCs REFERENCES intophotoreceptor-likecells,indicatingthatthecytoskeleton 1XieMS,XuGX,HuangY.RatModelforAnteriorSegmentIntraocularSurgery InducedBlood-RetinalBarrierBreakdown. 2008;222:42-47 polymerizesanddepolymerizescontinuouslywithvariations 2XieMS,XuGX.Culture Modulationofhumanleukocyteantigen inthecellularmorphology.Myosinincreasessignificantlyto moleculesandcostimulatorymoleculesonhumanretinalpigmentepithelium. supplyadditionalpowertoadjustthepolymerizationof 2008;222:48-52 actin.Inthepresentexperiment,cytoskeletalproteinssuch 3SasportasLS,KasmiehR,WakimotoH,HingtgenS,vandeWaterJA, aszyxin,NAG22,prelamin-A/Cisoform1precursor, MohapatraG,FigueiredoJL,MartuzaRL,WeisslederR,ShahK.Assessmentof -tropomyosin,tropomyosinalpha-4chainisoform1,and therapeuticefficacyandfateofengineeredhumanmesenchymalstemcellsfor 茁 cancertherapy. 2009;106:4822-4827 CK-1significantlydecreased.Thismayhaveresultedfrom 4AmadoLC,SaliarisAP,SchuleriKH,StJohnM,XieJS,CattaneoS,DurandDJ, theterminationofthekaryokinesisofmaturecellsandcell FittonT,KuangJQ,StewartG,LehrkeS,BaumgartnerWW,MartinBJ,Heldman cycleretardation,therebyincreasingmyosin.Theincreasein AW,HareJM.Cardiacrepairwithintramyocardialinjectionofallogeneic myosinexpressionlikelyregulatesactinpolymerization. mesenchymalstemcellsaftermyocardialinfarction. Basedontheresearchanalysis,thedownregulationor 2005;102:11474-11479 5ChoSR,KimYR,KangHS,YimSH,ParkCI,MinYH,LeeBH,ShinJC,Lim upregulationof proteinsenablesthe continuous JB.Functionalrecoveryafterthetransplantationofneurallydifferentiated polymerizationanddepolymerizationofthecytoskeleton mesenchymalstemcellsderivedfrombonebarrowinaratmodelofspinalcord duringBMSCdifferentiation.Thisbehaviorleadstothe injury. 2009;18:1359-1368 changeinthecellularmorphologyoftheBMSCsfromthe 6LadakA,OlsonJ,TredgetEE,GordonT.Differentiationofmesenchymalstem initialfusiformintothespindle-shapedphotoreceptor-like cellstosupportperipheralnerveregenerationinaratmodel. 2011; cells.Studieshaveshownthatmechanicaltensionisusedto 228:242-252 7AbdallahBM,KassemM.Humanmesenchymalstemcells:frombasicbiologyto controlthedifferentiationofstromalstemcellsthroughthe clinicalapplications. 2008;15:109-116 [41] actinmicrofilamentcomplex .Theproteinsareclosely 8PittengerMF,MackayAM,BeckSC,JaiswalRK,DouglasR,MoscaJD, relatedtothehighlydiversebiologicalactionofcells, MoormanMA,SimonettiDW,CraigS,MarshakDR.Multilineagepotentialofadult stabilization, and signal conduction.However,further humanmesenchymalstemcells. 1999;284:143-147 researchisneededtodeterminetheirmechanismsandtheir 9WilsonA,ShehadehLA,YuH,WebsterKA.Age-relatedmoleculargenetic changesofmurinebonemarrowmesenchymalstemcells. 2010; interactions. 11:229 Insummary,thedifferentialproteinprofileof 10DominiciM,LeBlancK,MuellerI,Slaper-CortenbachI,MariniF,KrauseD, photoreceptor-likecellsfromBMSC-induceddifferentiation DeansR,KeatingA,ProckopDJ,HorwitzE.Minimalcriteriafordefining wasestablishedby adoptingadifferentialproteomics multipotentmesenchymalstromalcells.TheInternationalSocietyforCellular method.Atotalof32differentiallyexpressedproteinswere Therapypositionstatement. 2006;8:315-317 identified,amongwhich11wereupregulatedand21 11HalfonS,AbramovN,GrinblatB,GinisI.Markersdistinguishingmesenchymal stemcellsfromfibroblastsaredownregulatedwithpassaging. proteinsweredownregulated.Thepresentstudyprovides 2011;20:53-66 importantinformationfordeterminingtheexactmechanism 12BoucherSE.SimplifiedPCRassayfordetectingearlystagesofmultipotent ofdifferentiationofBMSCsintophotoreceptor-likecells. mesenchymalstromalcelldifferentiation. 2011;698:387-403

473