高糖介导入牙周膜细胞凋亡中bcl-2、bax的作用及机制（英文）.pdf

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中国组织工程研究第18誊第57期2014—12—10出版ChineseJournalofTissueEngineeringResearchDecember10,2014VoL78．No．51wlA／vv．CRTER．orgRoleandmechanismofbcl-2andbaxinhighglucose--mediatedapoptosisofhumanperiodontalligamentcellsRenWei．wei’ChenShu．1an，QiuJing，LuShu．1ai，LiuHal—rong，LiuShi-hal。，1CollegeofStomatology,WeifangMedicalUniversity,Weifang261000,ShandongProvince，China2DepartmentofStomatology,QingdaoMunicipalHospital(Ora1)，Qingdao266071，ShandongProvince，China3CentralLaboratory,AfiliatedHospitalofQingdaoUniversityMedicalSchool,Qingdao266555,ShandongProvince，ChinaAbstractBACKGR0UND：Highglucosecantriggertheapoptosisofhumanperiodontalligamentceils．inwhich，whetherRenWei-wei，Studyingforbck2andbaxarestarted?Howdotheywork?Thereisst⋯norelevantreportmaster’sdegree，CollegeofStomatology,WeifangOBJECTIVE：Toinvestigatetheefectofhighglucoseonbcl-2andbaxmRNAexpressionaswellasapoptosisMedicalUniversity,Weifanginhumanperiodontalligamentcells／nvitro．261000，ShandongProvince，METH0DS：Humanperiodontalligamentcelswereprimarilyculturedandidentified．Then．cellsat5—8Chinapassageswereselectedinthisexperiment．Celsweretreatedwith5．5mm0I／L(controlgroup)and25mmol／L(highglucosegroup)glucosefor24and48hours．CellapoptosiswasdeterminedbyHoech~33258stainingCOrrespOndingauthor：Chenandtheexpressionofbck2andbaxmRNAwasdetectedbyReal-timePCR．Shu．1an．AssociatechiefRESULTSANDCONCLUS10N：Highglucose(25mmol／L)couldInduceIheapoptosisofhumanperiodontalphysician，Associateligamentcellsandincreasebcl-2andbaxsignificantly．Thebck2／baxratioshowedamoresignificantdeclineinprofessor,Master’ssupervisor,DepartmentofthehighglucosegroupcomparedwiththecontrolgroupfP<0．05)．Thesefindingsindicatethathighglucosecaninducetheapoptosisofhumanperiodontalligamentcels，inwhich，Bcl一2familyplaysanimportantrole．Stomatology,QingdaoMunicipalHospital(Ora1)，Qingdao266071，ShandongSubjectheadings：periodontalligament；diabetesmellitus；apoptosis；genes，bcl一2Province，ChinaFunding：theProjectofScienceandTechnologyBureauofQingdao，No．2012—1—3-1-(15)一6shdoi：l03969／]issn20954344RenWW，ChenSL，QiuJ，LuSL，LiuHR，LiuSH．Roleandmechanismofbcl一2andbaxinhigh2O1451O11glucose—mediatedapoptosisofhumanperiodontaligamentcels．ZhongguoZuzhiGongchengYanjiulhttp：／www．crterorg】2014；18(51)：8254—8260．Accepted：2014-10-28INTRODUCTlONPeriodontaldiseasesrefertoaseriesofcommonperiodontalligamentcellstodiseasesafectingthehealthofperiodontalcontinuouslyformnewprimaryfibersandtissue，whicharecausedbyperiodontalcementumaswellastoremodeIthealveolarpathogenssuchasPorphyromonasgingivalisbone．PeriodontalligamentcellsshowgoodAeromonas．Prevotellaintermedia，丁annereflafunctionsintissueregenerationandfunctionalforsyth／a。andAggregatibscterrecovery,whichplayavitalroleininflammatoryacfinomycetemcomitans．TheseinfectionscanperiodontaltissueIeadtooraltissuedestruction．toothdislocationandlooseoftooth”J．Currently,patientswithDiabetesmellitusisametabolicdiseasesevereperiodontitisinthepopulationaccountscharaclerizedbyhighbloodglucose．f0r10％一15％．evenupto30％．PeriodontitisPersistenthighbloodglucosecanIeadtoisacommonhumanoraldisease．andalsothechronicdamageanddysfunctiontoavarietyofmainreasonforadulttoothlOSS．tissuesJ．Thereisaclosetiebetweenperiodontaldiseaseanddiabetesmellitus．andSusceptibilitytoperiodontitisisdramaticallytheformeroneisregardedasthesixthlargestvaryinginthepopulation，dependingonhost’speriodontaIcomplicationsofdiabetesreactionstoperiodontalpathogens．Althoughmellitusl．Ontheonehand．diabeticpatientsperiodontaIdiseaseisdirectlycausedbyrelativelyhavehighprevalenceofperiodontaIdiseasethatisdificuIttotreat[9-10】：ontheotherbacteriaIplaque．inducedinflammation．thehosthealthandgeneticfactorshaveagreathand．．fglucosemetabolismhasbeenimpactontheclinicalmanifestationsandeffectivelycontrolled．theimprovementinprogressionofperiodontaldiseaseperiodontaldiseaseisobvious．OutcomemeasuresfortwodiseasesalsohavesomePeriodontalligamentcellsarethemostconnection，forexample，correlationbetween8254P．O．Box10002,Shenyang110180www．CRTER．org Reneta1．Roleandmechanismofbcl-2andbaxinhighglucose-mediatedapoptosisofhumanperiodontalligamentcellswww．CRTER．orgglycatedhemoglobinandperiodontalindicesandcorrelationmedium．Thesubsequentoperationswerecarriedoutinabetweenplasmalipidperoxide(oxidativestressindex)andsupercleanbench．Theteethwererinsedwith0．01mol／Lperiodontalmarkers⋯．1tsmechanismmaybethathiqhPBScontaining1000U／Lpenicillinand1000U／Lsugarinhibitsperiodontalligamentcellproliferation，andstreptomycin．AsterilescalpeIbladewasusedtoscrapeinducescellapoptosisaswelIasimpactsrelatedgrowththeperiodontiumatmiddle1／3oftheroot．andthefactorsrelated．specimenswereplacedintoPBScontaining0．3g／Llcollagenaseat37。Cwaterbathfor0．5houroscillationApoptosisisanactiveandorderedcelldeathprocessunderdigestion，gentlypipettedandcentrifuged(1000r，min×genecontrol，whichisabasicphysiologicalactivityfor5minutes)．AfterremovaIofthesupernatant．thecellpelletmulticellularorganismstoremovedamagedandharmfulwasculturedinaMEMmediumcontaining20％fefaIcells【1．Ifthisphysiologicalactivityisabnorma1．manybovineserum，resuspended，andcentrifugeddiseasescanoccur．Bcl一2familyisagenefamilycontrollingf1000r／minx5minutes)．Aftersupernatantremoval，acellapoptosis[⋯51．whichcanbedividedintoanti．apoptoticsmallamountofaMEMmediumcontaining20％fetaIgenes(suchasbcl-2)andpro-apoptoticgenes(suchasbovineserumwasaddedandgentlypipetted．Thebax)．Highglucosecaninduceapoptosisofhumansuspensionftissueblockscontainingasinglecellandperiodontalligamentcells。inwhich．whetherbcl-2andbaxundigestedones)wasseededincultureflasks，andtherecanbestarted?Howdotheywork?Thereisnorelevantwasanintervalofabout5mmbetweentissueblocks．Thereport．Thisstudyaimedtoexplorethemechanismofactionflaskswerereversed．and3mLofa—MEMmediumofbcl-2andbaxbystimulatinghumanperiodontalligamentcontaining20％fetaIbovineserum．100U／Lpenicillinandcellsbyhighglucose．1O0U／Lstreptomycinwasadded．Then．thecultureflaskswereplacedinaC02thermostatincubator(5％C02，MATERIALSANDMETHODS100％humidity,37。C1instaticculture．FourhoursIater，Designthecultureflaskswerereversedagain，andthebottomAsinglesampleobservation．wasdownward．Themediumwasexchangedevery4days．andundertheinvertedmicroscope．cellTimeandsettingextravasationandgrowthwereobserved．WhenthetissueTheexperimentwascompletedintheCentralLaboratoryblockwascoveredwithcellsand30％cellswere{ntheoftheAffiliatedHospitalofQingdaoUniversityMedicalculturebottles，cellsweresubcultured．Schoolfr0mJanuarytoJune2014．1mmunohistochemicalstainingwasemployedtodeterminecelIsource．andcellsatpassages5—8wereMaterialsusedintheexperiment．Reagentsandequipmentsforbcl-2andbaxinhighglucose—mediatedapoptosisofhumanperiodontalHoechst33258fluorescentstainingofhighglucoseligamentcellsareasfollows：andlipopolysaccharideefectsonhumanperiodontalligamentcellapoptosisAccordingtothepreviousreferencesandReagentsandinstrumentspre．experimentaIresults．thereweretwogroups}ntheReagentandinstrumentSourcestudy，physiologicalcontrolgroupwithaglucoseFelalbovineserumGibco，USAconcentrationof5．5mmOI，L．highglucosegroupwithaMEM，DMEMculturemedium，trypsinHyclone，USA25mmOI儿．OfDMEMmediumasinterventionreagent[’。18】_Cellsatadensityof1×10perwellwereHoechststainingkitBeyotimeBiotechnologyseededinto24．wellcultureplates．andaMEMcultureHangzhou，Chinamediumcontaining10％fetaIbovineserumwasadded．FastStartEssentiaIDNAGreenMasterKitRoche，SwitzerlandWhenthecelldensityreachedabout70％．theculture3111typeincub~orThermOFOrma．USAmediumwasremovedandcellsweresubjectedtoDMILinvertedphasecontrastmicroscopeLeica，Germanystarvationcultureinserum．freeaMEMmediumfor12hours．Accordingtothegrouping，twokindsofLightCycler96Real—timeFluorogenicRoche，SwitzerlandinterventionaIreagentswereadded，respectively，threeQuantitativePCRwellsineachgroup．for24and48hours．Afterlhat，theMethodsculturemediumwasaspirated．0．5mLfixativeperwellCultureandidentiflcationofhumanperiodontalwasadded．After10minutes．thefixativewasremovedligamentcellsandtheplateswererinsedwithPBStwice，3minutesTissueexplantmethodandenzymaticdigestionmethodonce．Then，0．5mLHoechst33258dyeliquorwasaddedwereused【‘Specimenswereprovidedbythefor5minutesdyeing。andtheplateswereshakenDepartmentofStomatology,QingdaoMunicipalHospitalseveraItimesbyhand．Thedyeliquorwasaspirateddyeandinformedconsentwasobtainedfr0mchildrenandandtheplateswerewashedwithPBStwice，observedtheirparents．HealthpremolarteethwereextractedfrOmandphotoedunderthefluorescencemicroscope．The11—14一year-oldpatientsbecauseoforthodonticremova1．excitationwavelengthwas350nm．andtheemissionandplacedimmediatelyintoaprecooledsterileaMEMwavelengthwas460nm．tSSN2095—4344CN21-1581，RcoDEN：zLKHAH8255 RenWW,eta1．Roleandmechanismofbcl-2andbaxinhighglucose-mediatedapoptosisofhumanperiodontalligamentcellsWWW．CRTER．0rg尺eaI-timePC尺detectionofbaxandbcl-2expressionsRESUL_TSTwo．stepRT．PCRwasadoptedinthestudy．MorphologyandsourcesofhumanperiodontalHoechst33258fluorescencestainingwasdoneasabove．ligamentcellsAfterthat．0．5mLTrizoIperwellwasaddedat15—30。CAfter4—7daysofculture．hun1anperiodontalligamentcellsstandingfor5minutes，sonucIe0pr0teincouldbefullywerefoundswimfr0mthesurroundingtissueblock．Cellsdissociated．After0．1mLchloroformwasadded．thewereextendedtoformastellateorfus~ormshapeandplateswerefullyshakenvigorouslyfor15seconds．interconnectedtoformamesh．Sporadiccellswerevisibleinstandingat15—30℃for2—3minutes．foIlowedbyanemptyareawithnotissueblock．WiththeincreaseofcelI12000r／mincentrlfuaatiOnat4℃for15minutes．density,cellswerespindle-shapedandexhibitedatissueCentrifugedsampleswerelayered．andRNAwasfoundblock-centeredarrangementinaradialappearance．intheupperaqueousphase．ThesupernatantwasImmunohistochemicaIstainingshowedthattheanti-vimentintransferredtoanewtube，addedwith0．3mLexpressionpolyclonalantibodyexpressedpositively,theisopropano1．mixedbyinversionandstoodat15—30℃cytoplasmwasbrownincolor,andanti—keratinexpressionwasf0r10minutes．Then．somegelatinousprecipitatesnegative(Figure1)．．appearedonthebottomofthetube。namelyRNA．TheRNAsampleswerecentrifugedat4℃at12000r／minResultsOfHoechst33258stainingfor10minutes。andthesupernatantwasdiscarded．TheNuclearcondensation，fragmentation。andappearanceofretainedprecipitatewasaddedwith0．5mLOf75％apoptoticbodiesareimportantcharacteristicsofcellethanolcontaining0．1％DEPCandmixedgently．A矗erapoptosis．Underthefluorescencemicroscope，apoptotic12000r／mincentrIfugatiOnfOr5minutesat4℃，thecellshadstainedanddensenucleilnthecolorofbrightsupematantwasremoved．TheRNAsamplesweredried10white，andnormalcells，roundoroval，showedauniformminutes，anddissolvedin30UL0fDEPCat55—60℃f0r10bluefluorescencewithclearnucleusboundaries．Apoptoticminutes．Afterwards，theA26o，A280ratiowasmeasured，cellswererareinthenormaIcontroIgroup．whileapoptoticTranscriptorFirstStrandcDNASynthesisKitwasusedcells，evenapoptoticbodies，werevisibleinthehighglucosef0rRNAreversetranscriptionandsynthesisofcDNA．group．ThenumberofapoptoticcellsorapoptoticbodiesPrimerdesignbasedonthecodingregionisshowninwasincreasedsignificantlyinthehighglucosegroupwithTable1．time(Figure2)．mRNAexpressionofbcl-2andbaxTable1Expressionofbax，bcl-2andGADPHmRNAComparedwiththecontroIgroupf5．5mmol／L)，thebcl-2andbaxexpressionlevelsinhumanperiodontaIligamentcellsweresignificantlyincreasedinthehighglucosegroup(25mmol／L1at24and48hours(P<0．05)．Thebcl-21baxratio，however，wassignificantlylowerinthehighglucosegroupthaninthecontrolgroup(P<0．05；Figure3)．DISCUSSIONReal—timePCRwasdonereferringtotheinstructionsofDuetothefrequentphysiologicalactivities，suchaschewRocheFastStartEssentiaIDNAGreenMasterKit．Theandlanguage，periodontaltissueremodelingisveryactive．reactionsystemwas2OUL，andtemperatureprogramPeriodontalligamentcellsareoneofthemostimportantasfoIlows：10-minuteactivationofFastStartTaqDNAcellsintheperiodontaIIigamenttissue．whichplayanpolymeraseandDNAdenaturationat95℃．ignoredroleinperiodontaltissueregenerationandnewAmplificationprogram：95℃for10seconds．60℃forattachmentformation．Undertheinterferenceofsome10seconds，72℃for20seconds，totally45cycles．baxvirulencefactors．thebiologicalactivityofhumanperiodontalbcl-2，andGAPDHwereamplifiedsimultaneously．ligamentcellsisinhibited，therebyafectingthenormalTargetgeneexpressionwascalculatedaccordingtofunctionoftheperiodontalIigamentthatcaneventuallyIead2⋯method．toperiodontaldiseases．MainoutcomemeasuresClinicalandepidemiologicalsurveyshavefoundahighIdentificationofmorphologyandsourcesofhumanincidenceofperiodontitisindiabeticpatients．withseriousperiodontalligamentcells；observationunderconditionandrapidprogress：ontheotherhand。afterthehoechst33258fluorescencestaining；bcl-2。baxgeneefectivecontroIofbloodglucose．periodontaIdiseasehasexpressions．alsobeenimprovedsignificantly．1nbasicresearch．theefectsofhighglucoseonhumanpedodontaIIigamentcellsareStatisticalanalysismanifoId，mainlyintermsofproliferation，collagensynthesis，Thedatawereexpressedasmean+SD，andanalyzedbytdiferentiation．andhostimmuneresponse．testusingSPSS19．0software．AvalueofP<0．05wasconsideredsignificant．Cellproliferationisthebasisofbiologicalgrowth8256户o．Box10002,Shenyang110180vv~fw．CRTER．org Ren~eta1．Roleandmechanismofbcl-2andbaxinhighglucose-mediatedapoptosisofhumanperiodontalligamentcellswww．CRTER．org．．．．．．．．——————developmentandreproduction．HighglucosecanNumerousstudieshaveindicatedthattheaggravationofremarkableinhibitthe／nvitroproliferationofhumandiabetesmellituscancauseanincreaseininvivoperiodontalligamentcells．KimetalIltlJfoundthatwheninflammatoryfactorsandendotoxinlevelsaswellashosttheglucoseconcentrationis25mmol／L．celIproliferationresponses．andthenworsenchronicperiodontitis[2科．canbesuppressed．Basicfibroblastgrowthfactorisaheparin-bondedpeptidedirectlyinvolvedincelIdivisionHighglucoseincreasesgeneexpressionofinflammatoryandproliferationtopromotebloodvesselgrowthandfactors．Astudyfr0mFangfoundthathighglucoseitselfdevelopment．Basicfibroblastgrowthfactorisassociatedcaneitherelevate1nterleukin一113，interleukin．6。tumorwiththeproliferationandmigrationofhumanperiodontalnecrosisfactor-ageneexpressions，orenhancetheligamentcellsinvitro0highglucoseinhibitssecretionofadvancedglycationendproducts．Co—existenceofhighbasicfibroblastgrowthfactor~Ohgiandco．workers[z~JglucoseandinflammatoryfactorscanleadtomoreseverediscoveredthatbasicfibroblastgrowthfactorexpressionIocaIdamagetotheperiodontaltissue．isdecreasedwiththeincreasingglucoseconcentration(5．5mmol／Land20mmol／L)．ExperimentalfindingsToll—likereceptors(TLR)areaclassofimportantproteinindicatethathighglucosecanweakenthecelIdivisionmoleculesinvolvedinnonspecificimmunity'butabridgeandproliferationbyinhibitionofbasicfibroblastgrowthbetweennon-specificandspec~icimmunities．TLR4existsfactorinfibroblasts．macrophagesandepitheliaIcellsfromtheperiodontaltissue．ItisspeculatedthathighbloodglucoseCollagenisthemaincomponentoftheperiodontalIigament．cancontributetotheexpressionofthesereceptorsinthewhichguaranteesthestructurestability,normalfunctionandperiodontaItissuesandjreprovethecontentofthephysiologicregenerationoftheperiodontaltissue．Highcorrespondingligand，thusaccumulatingligandcomponentsglucoseinhibitscollagensecretion．Changetal【JinterferedStudieshaveshownthat．eitherchemicaIormechanicaIpenodontalligamentcellswithhighglucoseandperiodontaItreatmentscanimprovebloodglucoseandIipid1ipopolysaccharide，andtheyfoundthatonlylevels[。≈m．Changetal[21undthathighglucosecanhigh—concentrationglucosecouldsignificantlyinhibitbothenhancetheeffectoflipopolysaccharideontheexpressiontypeIandtypelVcollagenexpressionlevels．0fTLR2inperiodontalligamentcells．Underthestimulationofcertainfactors．periodontaIligamentApoptosisisaprogrammedcelldeathprocessstimulatedbycellswithavarietyofbiologicaIfunctionscandiferentiategeneregulationundercertainconditions．Innormaljntoosteoblast—likecells．Highglucosecanreducethephysiologicalactivities，apoptosisplaysanactiveroleinosteogensisandmineraIizatiOncapacityofperiodontalclearingaginganddamagedcells，respondingtoIigamentcells。KimetalI‘纠observedwhentheperiodontaIenvironmentaIchangesinvitroandinvivo．maintainingtheIigamentstemcellswereexposedtohighglucosehomeostasisinthebody．Thisimbalanced(30mmol／L)，areductionwasfoundinosteoblastactivity．microenvironmentmaybeassociatedwithmanydiseases．ScleraxisisamemberofTWistsubfamilyincel1．specificIncreasingstudieshavesuggestedthatcellapoptosisisbasichelix-loop—helixtranscriptionfactor,closelyrelatedtoinvolvedintheoccurrenceanddevelopmentofthediferentiationofconnectivetissue．Highglucosecanperiodontitis．HighglucosecanfunctionnormallybyincreasemRNAexpressionofScleraxisbutinhibitinducingapoptosisofperiodontalligamentcells．JiaqiangetosteogenicdiferentiationofperiodontalIigamentcells．a，foundthatwhen25mmollLglucosecaninduceYuanetal‘。detectedScleraxismRNAexpression：napoptosisinhumanperiodontalligamentcellsinahumanperiodontalIigamentcellswassignificantlyconcentration—dependentmanner．increasedunderhigh．glucoseconditions．andalkalineph0sDhataseactivitydecreased．ExperimentaIfindingsHoechst33258stainingisacommonmethodforindicatethathighglucoseraisesScleraxisexpression。observationofapoptoticmorphology,characterizedastherebyinhibitingosteogenicdiferentiation．Kimetal0IoJIntuitiveobservationofapoptosisandsimpleandfastfoundthat，underhigh—glucoseconditions．theoperation，therebydeterminingwhetherthecellapoptosismineraIizatiOnpercentageofperiodontaIIigamentcellsoccursandprovidingagoodreferenceforsubsequentwasIowerthanthatundernormaIconditions．experiments．After24hoursofhighglucosestimulation，periodontalligamentcellscanbefoundinanincreaseofBacteriaIactivity-activatedhostimmuneinflammatoryapoptoticcells．chromatinc0ndensatiOnphenomenonresponseisconsideredtobeanimportantpartofsoftandoccurs：andapoptoticbodiesappearafter48hours．hardtissuedamageinperiodontaldiseases．SomeinfIammatorycytokines．includingintedeukin．1，interleukin-6MitochondriaarethecentrelJinkintheregulationofandtumornecrosisfactor-a。mayInducedamagetotheapoptosis．andthemechanismisassociatedwithBcl-2connectivetissueandalveolarbone．Highexpressionsofproteinfamilyinteractions．changesinthemitochondriaItheseinfiammatorycytokinesarefoundinthegingivalmembrane．intracellularCaoverloadandoxygenfreesulcusfluidofpatientswithgingivitisandperiodontitis，radicals卜)．Bc1．2familyproteinsareIocatedinthesuggestingthatelevatedlevelsofthesecytokinesmayexertmitochondrialmembraneandcanleadtoapoptosisdirectlyanimportantroleinperiodontaItissuedamage．orbyactivatingCaspasepathway,whichcanbedivided|ssN2095_4344cN21-1581／RcoDEN：zLKHAH8257 璺!竺|：坠in_h|ghglucose-mediacedap0plos{sofhumanperiodontalfigamentcelswww．CRTER．orghigh-glucosefoodsisthebasicmeasuretoreducetheserisk【13】NobleBS，StevensH，LoveridgeN，eta1．Identificationoffactors．Nutritious．high-fiberfoodssuchasfruitsandapoptoticchangesinosteoc~esinnormaIandpathologicalvege~blescankeepalotofhealthbenefits[~uj．(2)Toimprovehumanbone．Bone．1997；20(3)：273．282．chewingperformance．FulIorpartialremovabledentures【14】SuzukiH，WildhirtSM，DudekRR，eta1．InductionofcannotbecomparabletonatureteethinterrT1s0fchewingapoptosisinmyocardialinfarctionanditspossiblerelationshipefficienc：however,thefixedpartialdenturesandimplanttonitricoxidesynthaseinmacrophages．TissueCel1．1996；denturesarehelpfultorestorechewingperformanceand28(1)：89—97．improvedietstructure．Fordoctom：theoFalcomplicationsof【15】SzabolcsM，MichlerRE，YangX，eta1．ApoptosisofcardiacdiabetesmellitusmainlyincludeperiodontaIdisease．myocytesduringcardiacallograftrejection．Relationtocandidiasis，bumingmouthsyndrome，andacuteorelinductionofnitricoxidesynthase．Circulation．1996；94(7)：infections．A¨thesediseasesarebasicallytobefoundand1665．167&treatedindentaltreatment．Therefore．thedentistsplaysa【16】蒋俊强，工忠朝，黎春晖，等．三种方法原代培养人牙周膜细胞【J】lfundamentalroleinthediagnosis．treatmentandcontroJof中国组织工程研究与临床康复，2010，14f23)：4290．4294．metabolicpatientsJ．Inthefuture．ahigherlevelof【17】YuanYD，MiaoS，XieH．EfectofhighglucoseonthemultidisciplinarycooperationbetweendentistsandexpressionoftranscriptionfactorScleraxisinperiodon~lendocdnologistsisrequiredtoimplementthetreatmentofligamentcellsinvitro．ZhonghuaKouQiangYiXueZaZhi．metabolicsyndromeandjtscomplications．2008；43(11)：668-670．【18】KimHS，ParkJW，YeoSI，etaI，EffectsofhighglucoseonREFERENCEScelularactivityofperiodon~lligamentcellsinvitro．Diabetes【1】AndriankajaOM，SreenivasaS，DunfordR，eta1．ResCI．nPract．2006；74(1】：41—47．Associationbetweenmetabolicsyndromeandperiodontal【19】TanZ，GongP，ZhaoQ．Influenceofbasicfibroblastgrowthdisease．AustDentJ．2010；55：252—259．factorsonthemigration，proliferationofosteoblastsand【2】LuiJ，WuDingeta1．Evaluationofserumlevelsofperiodontalligamentfibroblasts．HuaXiKouQiangYiXueZaC-reactiveproteinandlipidprofilesinpatientswithchronicZhi．20O5：23(3)：201．203．periodontitisand／orcoronaryheartdiseaseinanethnic[20】OhgiS，JohnsonPW．GlucosemodulatesgrowthofgingivalHanpopulation．QuintessenceInt．2010；41：239·47．fibroblastsandperiodon~lligamentcels：correlationwith【3】ArmitageGC．Diagnosisofperiodontaldisease．Jexpressionofbasicfibroblastgrowthfactor．JPeriodontRes．PeriOdOntO10g2003；74：1237—1247．1996；31：579—588．【4】OrcianiM，TrubianiO，VigniniA，eta1．Nitricoxide[21】ChangPC，ChienLY,ChongLYeta1．Glycatedmatrixproductionduringtheosteogenicdiferentiationofhumanup-regulatesinflammatorysignalingsimilarlytoperiodontalligamentmesenchymalstemcells．ActaPorphyromonasgingivalislip0p0Iysaccharide．JPeriodontalHistochemica．2009；111(1)：15—24．Res．JPeriodontalRes．2013：48(2)：184．193【5】BjellandS，BrayP'GuptaN，eta1．Dentists，diabetesand[22】KimSLeeJY,ParkYD，eta1．Hesperetinalleviatestheperiodontitis．AustrDentJ．2002；47：202-207．inhibitoryeffectsofhighglucoseontheosteoblastic【6】MustaphaIZ，DebreyS，OladubuM，eta1．Markersofdiferentiationofperiodontalligamentstemcels．PLoSOnesystemicbacterialexposureinperiodontaldiseaseand2O13：8(6)：e67504．cardiovasculardiseaserisk：asystematicreviewand【231DinarelloCA．Immunologicalandinflammatoryfunctionsofmeta-analysis．JPeriodonto1．2007；78(12)：2289—2302．theinterleukin一1family．AnnuRevImmuno1．2009；27：【7】SandbergGE，SundbergHE，FjellstromCA，eta1．Type2519．550．diabetesandoralhealth-acomparisonbetweendiabetic[24JWongRK，Pet[itAI，QuinnPA，eta1．Advancedglycationendandnondiabeticsubjects．DiabResandClinPract．2000；productsstimulateanenhancedneutrophilrespiratoryburst50(1)：27-34．mediatedthroughtheactivationofcytosolicphospholipaseA2f8】TaylorGW，BorgnakkeWS．Periodontaldisease：andgenerationofarachidonicacid．Circulation．2003；108：associationswithdiabetesglycemiccontroland1858—1864．complications．OralDis．2008；14(3)：191—203．[25】AlenEM，MattewsJB，O’HalloranDJ，eta1．Oxidativeand【9】LallaE．Periodontalinfectionsanddiabetesmellitus：wheninflammatorystatusintype2diabetespatientswithwillthepuzzlebecomplete?JClinPeriodont．2007；34(11)：periodontitis．JClinPeridonto1．2011；38：894-901913．916．【26】PreshawPM，AlbaAL，HerreraD，etaIPeriodontitisand【10】刘洪臣，布静秋．老年人常见的El腔疾病[J】．中华老年口腔医学diabetes：atwowayrelationship．Diabetologia．2012；55：杂志，2008，6(4)：10018—10020．21—31．【11】1wamotoNishimuraF，NakagawaM，eta1．Theefectof【27】房明．高糖、糖基化终产物刺激下人牙周膜成纤维细胞生物学antimicrobialperiodontaltreatmentoncirculatingtumor性能变化的体外研究[D】西安：第四军医大学，2013necrosisfactor-alphaandglycatedhemoglobinlevelin[28]KatzJ，Bhallacharyyal，Farkhondeh-KishF，eta1．patientswithtype2diabetes．JPeriodonto1．2001：72：Expressionofthereceptorofadvancedglycationend774．778．productsingingivaltissuesoftype2diabetespatientswithf12】GrossiSG，SkrepcinskiFB，DeCaroeta1．Treatmentofchronicperiodontaldisease：astudyutilizingperiodontaldiseaseindiabeticsreducesgtycatedimmunohistochemistryandRT-PCR．JClinPeriodonto1．hemoglobin．JPeriodonto1．1997；68：713-719．2005：32：40—44．1SSN2095I4344cN21—1581，Rc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RenwW．ela1．Roleandmechanismofbcl-2andbaxinhighglucose-mediatedapoptosisofhumanperiodontalligamentcellswww．CRTER．orgf291D’AiutoF，ParkarM，AndreouG，eta1．Peri0dOntⅢsand【34】KorsmeyerSJ．Bcl-2genefamilyandtheregulationofsystemicinflammation：controIofthelocaIinfectionisprogrammedcelldeath．CancerRes．1999；59(7Supp1)：associatedwithareductioninseruminflammatorymarkers．J1693s-1700s．DentRes．2004；83：156-16O．【35】KawadzadehA，BourauelC，GotzW，eta1．Earlyresponsesof『301Navarro—SanchezAB，Faria-AImeidaR，Bascones-MartinezAperiodontalligamentcellstomechanicalstimulusinvivo．JEfectofnon·surgicalperiodontaltherapyonclinicalandDentRes．2005；84(10)：902-906．immunologicalresponseandglycaemiccontrolintype2【36】Nowjack-RaymerRE1SheihamA．Associationofedentulismdiabeticpatientswithmoderateperiodontitis．JClinanddietandnutritionInUSadults．JDentRes．2003；82：Periodontol_2007；34：835．843．123．126．【31】GanonalJ，BasconesA，AcevedoA，eta1．Apoptosisin【37】KrallE，HayesC，GarciaR．HowdentitionstatusandchronicadultperiodontitisanalyzedbyinsituDNAbreaks，masticatoryfunctionafectnutrientintake．JADA．1998；129：electronmicroscopy,andimmunohistochemistry．J1261．1269．Periodontol_2001；72(4)：517-525．【38】MoraisJA，HeydeckeG，PawliukJ，eta1．Theefectsof【32】LiuJ，WuWangB，eta1．Highlevelsofglucoseinducedthemandibulartwo．implantoverdenturesonnutritioninelderlyCaspase-3／PARPsignalingpathway,leadingtoapoptosisinedentulousindividuals．JDentRes．2003；82：53-58．humanperiodontaIligamentfibroblasts．CelIBiochem【391VernilloDiabetesmellitus：relevancetodentaltreatment．Biophys．2013；66(2)：229-237．OraISurgOraIMedOraIPathoIOraIRadioIEndod．2001；91：【33】SchaferB，QuispeJ，Choudhary、，'eta1．Mitochondrialouter263．270．membraneproteinsassistBidinBax-mediatedIipidicpore『401VernilloATDentaIconsiderationsforthetreatmentofpatientsformation．MoIBioICel1．2009：20(8)：2276—2285．withdiabetesmellitus．JADA．2003：134：24s．33s．高糖介导人牙周膜细胞凋亡中bcl-2、bax的作用及机制任伟伟’，陈书兰2仇静2卢恕来，刘海蓉2刘世海。(’潍坊医学院口腔医学院，山东省潍坊市261000；青岛市市立医院口腔科，山东省青岛市266071；。青岛大学医学院附属医院中心实验室，山东省青岛市266555)任伟伟，男，1978年生，湖北省十堰市(15)-nsh)对文章负责。人，汉族，潍坊医学院在读硕士，主要从利益窕文章及内容不涉及相关利事牙周病的基础及临床研究。摘要益冲突。通讯作者：陈书兰，副主任医师，副教授，背景：高糖环境下人牙周膜细胞会发生凋缨r要菘惠儿及其家属自愿捐献离硕士生导师，青岛市市立医院口腔科，山亡，其中bcl-2，bax是否启动?如何发挥体牙，对实验过程完全知情同意。东省青岛市266071作用?此方面尚未见有文献报道。爵牙周膜细胞一是牙周膜中目的：应用不同浓度葡萄糖影响人牙周膜最常见的细胞，不断形成新的主纤维、牙文章亮点：细胞，观察细胞凋亡及6cI-2、bax基因表骨质，并改建牙槽骨，对牙周组织的修复1文章首次从凋亡形态学角度观察了高达的变化。十分重要，是牙周治疗后形成牙龈与牙根糖介导人牙周膜细胞凋亡影像，首次利用方法：原代培养并鉴定人牙周膜细胞，取面新附着的主要细胞来源。Real—timePCR检测了人牙周膜细胞高糖5—8代细胞用于实验。采用5．5mmol儿者嘲：文章为原创作品，无抄袭诱导下bcl-2，bax表达。葡萄糖(生理对照组)和25mmoI，L葡萄糖剽窃，无泄密及署名和专利争议，内容及2实验结果表明，Hoechst33258免疫荧(高糖组)作用细胞24h和48h；以数据真实，文责自负光操作简单，影像清晰，可直观辨识细胞凋Hoechst33258免疫荧光染色观察人牙周亡情况。RT-PCR显示25mmol／L葡萄糖膜细胞凋亡；以Real-timePCR检测中图分类号：R318文献标识码：A促进人牙周膜细胞bcl-2、bax表达增强；bcl一2、bax表达。文章编号：2095—4344(2014)51—08254—07bcl-2／bax比值下降。提示高糖可增加人牙结果与结论：25mmol，L葡萄糖促进人牙周膜细胞凋亡，Bcl-2家族发挥了重要作用。周膜细胞凋亡，bc1．2、bax表达显著高于任伟伟，陈书兰，仇静，卢恕来，刘海蓉，关键词：对照组(P<0．O5)；bcl-2／bax比值显著低刘世海．高糖介导人牙周膜细胞凋亡中组织构建：高糖：人牙两貘缅匏：凋亡：于对照组(P<0．o5)。结果说明高糖可增加bcl一2、bax的作用及机制【J】l中国组织工程研8cl-2：Bax人牙周膜细胞凋亡，Bcl-2家族发挥了重究，2014，18(51)：8254—8260．主题词：要作用。牙疑骥：糖渌病；细瞻凋亡：基因．Bcl-2(EditedbyLiM，ChenLL／WangL)基金资助：者贺葡设计、实施、评估为本文寿岛发局(2012—7_3—7—作者，均受过专业培训，第一作者成文并8260Po．Box10002,Shenyang110180www．CRTER．org