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Different

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Different

    Different

    Feb.2008,Volume5,No.2(SerialNo.39)JournalofUS.ChinaMedicalScience,ISSN1548?6648,USA

    DifferentRolesofBDNF,KDRandBaxinLiverResponsetoAcuteor

    RepeatedImmobilizationStress:EvidencefromQuantifiedReverse

    Transcription.polymeraseChainReactionResearch

    WANGYutongi

    .

    WANGWen2

    .

    TANQing.rong2.XIABing3.DOUKe-fengi

    r,.DepartmentofHepatoandBiliar3'Surger3;XijingHospital,theFourthMilitao"MedicalUniversitk;Xi"an710032;

    2.PsychosomaticDepartment,XijingHospital,theFourthMilita~MedicalUniversit>;Xi"an710032;

    3.Regime,,CollegeofStomatolog3;theFourthMilitaryMedicalUniversio;Xi'an710032) Abstract:ObjectiveToinvestigatethemRNAlevelsforBDNFKDRorBaxintheJivertissuesfrom

    controlratsandthoseunderacute/single(3h/d)orrepeated/chronic(3h/dforl5days)immobilizationstress

    conditions.MethodsThequantifiedreversetranscription

    polymerasechainreaction(IPCR)wasemployed

    withthechronicimmobilizationstressratmode1.ResultsAcutestresssignificantlyincreasedthemRNAlevelfor

    KDR(1l4.8%ofcontro1).While.fifteenrepeatedimmobilizationssignificantlydecreasedthemRNAlevelfor

    BDNFf82.22%ofcontro1).Neitheracutenorrepeatedjmmobilizationchangedtheexpressi

on1eve1ofBfix.Six

    repeatedimmobilizationsinducedasignificantbodyweightlossandthiseffectkeptduringthewholeobserving

    window.ConcluslonThepresentdatasuggestedthatKDRorBDNFisinvolvedintheacuteorchronic

    immobilizationinducedliverresponse,respectively.LocallysynthesizedBaxisnotstronglyinvolvedintheliver

    responsetoacuteorrepeatedimmobilizationstress.

    Keywords:brainderivedneurotrophinfactor(BDN;kinaseinsertdomain

    containingreceptor(KDR);

    bax;immobilizationstress;reversetranscription

    polymerasechainreactionfRT-PCR);ratliver

    Stressjsoneofthemajorriskfactorswhichaccountsforthelncreasedlncidenceofanumberofcommon

    life

    threateningdisorders.Itisnowgettinghighlightinnotonlyproducingsomenervoussystemdependedmental

    problemssuchasdepression

    butalsoresultinginsomesubstantialstructurechangeoutsideofthenervous systemlikeinheartliveretc.Althoughtheimportanceofstressinlifeappearstobesignificant.thecellular

    andmolecularmechanismsinvolvedinthepath()physi()lOgyofstressremainslargelyunknownespeciallyfor

    thosenon

    neuronaltargetorgans.Thecaseforliverisevenworsethanthatforthecardiovascularsystemwhich

    receivedstrongfocusanddevelopedinarelativefasterspeed.Onlyrecentlyhasscientificevidencebeenreported

    todemonstratehowstressexacerbatesliverdiseases

4j.Therestilllackstudyonhowstressaffectthefunctionof

    normalliverandtheunderlyingmechanisms.Thepurposeofthisstudywastodetectthemolecularchangeof

    liverunderflmostcommonlyusedstressmode1.jmmobilization.

    StresscanalsoimpairHepaticBloodFlow(HBF)throughitseffectontheautonomicnervoussystem

    Acknowledgements:ThisworkwassupposedbygrantfromNationalNaturalScienceFoundationofChina(No.30500278)and

    theScientificProjectoftheFou~hMilitaryMedicalUniversity(No.05XJZ004). WANGYutong(1975

    ),male,Ph.D.candidateintheDepartmentofHepatoandBiliarySurgery,XijingHospital,theFourth

    MilitaryMedicalUniversity;researchfield:hepatoandbiliarydisease. 10

    DifferentRolesofBDNF,KDRandBaxinLiverResponsetoAcuteorRepeatedIm mobilizatiOnstress:

    !!!竺兰墅ReverseTranscription.polymeraseChainReactionResearch

    mdrectly?Thischangemayactivatetheliver'"feedbackreactiontocompensateforit .ThusDutthe

    kmase-insertdomain

    containingreceptor(KDR)atthecentralpointinthatKDRmediatesvascularendothelia1 growthfactor(VEGF)inducedAngiogenesis

    61.Baxisapro.apoptoticBc1.2familymemberthatp1aysacentra1

    roleintheregulationandmediationofapoptosisandalsointhecellularresponsetooxidativestress8.

    Accordingtotheabovementionedinformation,weemployedBDNF,KDRandBaxasthecandidate

    moleculeswhicharesuggestedtobeinvolvedintheliverresponsetoimmobilizationstress.Totestthedil.ferent

    effectofacuteandrepeatedimmobilization,weevaluatedthemRNAlevelforBDNEKDRandBaxinliver

    t1ssuealiertheratsreceivedoneacuteimmobilizationor15repeateddailyimmobilizationstresses

    .T.hecurrent

    studyindicatedthatBDNFandKDRaredifferentlyinvolvedintheacuteandrepeatedimmOb.1izatiOnstress

    mducedliveresponse?Tooursurprise,Baxseemsnottobeinvolvedinacuteorrepeatedimmobilizationstress

    inducedliverresponse.

    MATERIALSANDMETHODS

    1?ExperimentalAnimal

    Subjectswereadult(6_8weeks)maleSprague-Dawleyratsweighing200.250gatthebeginninofthe

    experiment.Ratswerecagedindividually7daysbeforetheimmobilizationsoastoavoidtheeffectofsocial

    contacts?Maleanimalswereusedtoavoidvariablesteroidlevelsseenduringthenormalestrouscycleoffemale

    animals?Animalshadfreeaccesswithfoodandwatera12:12

    hlight-darkcycle(1ightsonat06:00am).

    All

    testingprocedureswereapprovedbytheCommitteeofAnimalCareandUseforResearchandEducationatthe

    FourthMilitaryMedicalUniversity(Xi'an,RR.China)andeffortsweretakentoreducethesufferintotherats

    andthenumberofratsusedintheexperiment.

    2.ImmobilizationProcedures

    Wecarriedoutourrestrainedexperimentsatafixedtimebetween8:00and11:00am.Soastoinduce

    maximalstressresponseasreviewedintheliterature,thatthemaximumlevelofcorticosteron

eresponsesin

    animalsshouldbeprovokedduringthemorning.

    Forimmobilizationexperiments,ratswerekeptdailvfor3hin

    Plexiglastubes.Fouranimalswereusedforeachgroup(=4).Group1,unstressedanimals(contro1):group2,

    acutestress,single3hofstress;group3,chronicstress,3hofdailystressfor15days.Chronicstressedanimals

    werereturnedtotheirhomecageafterthedailyprocedure.RatswereweighedbeforetreatmentandeverY3days.

    15_30minafterthefinalstresstreatmenttheywereanaesthetizedwithsodiumpentobarbital(100mg/kgbody

    weight,i.pfollowedbyperfusiontranscardiallywith100mlnormalsaline

    .

    Controlmiceweresacrificed

    immediatelyaftertheremovalfromtheirhomecages.Livertissuewasrapidlyharvested,frozenandD0oledunder

    _

    80~CforfutureuseinRT-PCR.AllthesetreatmentsareblindtotheexperimenterwhoDerformedtheRT-PCR

    procedureuntilthefinalstageofanalyzing.

    3.RNAIsolationandcDNASynthesis

    TotalRNAwasextractedfromlivertissueaccordingtoapreviousprotocol[. Brieflv,afterthe

    mmobilizati0nprocedure,ratswereanaesthetizedwithsodiumpentobarbital(100mg/kgbodyweight,i.p.)and

    thenperfusedtranscardiallywith100mlNS.Livertissueswererapidlyharvested,frozenandpooled.TotalRNA

    wasextractedwithTrizol(GibcoBRL,USA),anRNAisolationreagent.ThefirststrandofcDNAwasreverse1v

transcribedwithM_MLVreversetranscriptase(GibcoBRL,USA)andoligo(dT)12

    18primer(Gibc0BRL.

    USA)byrandom

    primingat37"Cfor60min,andterminatingbyheatingat65~Cfor5min.Thesvnthesizedfirst 11

    DifferentRolesofBDNF,KDRandBaxinLiverResponsetoAcuteorRepeatedImmobilizationStress:

    EvidencefromQuantifiedReverseTranscription?polymeraseChainReactionResearch strandsofcDNAwereamplifiedaccordingtothefollowedPCRprocedure.

    4.ReverseTranscription.polymeraseChainReaction(RT-PCR)

    ThesynthesizedfirststrandsofcDNAwereamplifiedbyusingprimersspecificallydesignedforBDNE

    KDRandBax.

    actin.asoneofthe"housekeeping"genes.waschosenasanendogenousinternalcontroltowhich

    otherPCRamplificationproductswerenormalized.TheBDNF,KDR,Baxand

    actinprimersweredesigned

    accordingtotheirmRNAsequences,respectively.Thedetailedinformationoftheseprimersandannealing

    temperaturewasshowninTable1.PCRreactionswerecarriedoutin50glofreactionmixturecontaining10mM

    ofTris(pH8.3),50raMofKCI,1.5mMofMgC12,1"lofsynthesizedfirststrand,40pmolofeach5'and3'primer

    pair,0.1mMofeachdNTPand2.5unitsofTaqDNApolymerase(Takara,Japan).ThePCRreactionwas

    performedfor30cyclesbyusingaPTC

    100ProgrammedThermalController(MJResearch,USA)asfollows:

    denaturationat93~Cfor1min,annealingattheoptimaltemperatureshownintable1for30s,followedbyan

    extensionat72~Cfor8min.Allexperimentsincludedreversetranscriptionnegativecontrolswherereverse

    transcriptasewasomittedtoexcludethepossibilityofgenomicDNAamplification.Afteramplification,PCR

    productswereseparatedona3%agarosegelcontainingethidiumbromide.Forevaluationofchangesineach

    targetmRNA.gelbandswerevisualizedinaUV-transilluminatorandimageswerecapturedbyusingan8bit

    CCDcamera(Ultra

    VioletProducts,UK).Therelativegraylevelsofthebands,expressedasopticaldensityunits, werequantitativelyanalyzedbyusingLabworksImageSoftware(Ultra

    VioletProducts,UK).Inallcases,reverse

    transcriptionnegativecontrolswerenotamplifiedandcouldnotbedetectedonthege1.TargetmRNAexpression

    wasexpressedasaratioofdensitometricmeasurements(targetmRNA/

    actinmRNA).Theseratioswereaveraged

    andcomparedwiththosetakenfromcontrolsamples.

    Table1Informationofprimersusedinthestudy

    5.StatisticalAnalysis

    Resultsarepresentedasmean_S.E.M.andeachvaluerepresentanaverageof4animals.Datawereanalyzed

    usingone-wayanalysisofvariance(ANOVA).

    RESUIS

    1.RepeatedImmobilizationStressDecreasedtheRats'BodyWeight

    AsshowninFigure1.7daysbeforeandrightafterthefirstimmobilizationstress.thereisnodifierencein

    thebodyweightbetweenthecontrolandtheexperimentgroup.Atierthe3rdimmobilizationstress.theratshada

    slightlydecreasedbodyweight(91.15%ofthatofcontrolrats)butwithoutstatisticalsignificance.Theykept

    losingweightandthisdecreaseissignificantafterthe6"'immobilizationandkeptatthislevel(around88%ofthat

    ofcontro1)duringthel5day

    observingtimewindowfFig.1).IPCRfromnormallivertissueidentifiedDNA 12

    DifferentRolesofBDNF,KDRandBaxinLiverResponsetoAcuteorRepeatedImmobilizationStress:

    EvidencefromQuantifiedReverseTranscription-polymeraseChainReactionResearch productsforBDNF,KDRandBaxattheirexpectedsize,respectively(Fig.2). 104

    100

    80

    

    96.3O3691215

    Immobilizationcourse(day)

    Fig.1Bodyweightchangeduringtherepeatedimmobilizationstress

    Notes:Thebodyweightofcontrolratsandratsreceivingdailyrepeatedimmobilizationstresswasmeasuredon7and0,3,6,9,

    12orl5dafterstress.Thefirststresswasgivingonday0.Themeanbodyweightofstressedratswasexpressedaspercentageofthat

    fromtheircontrolcounterpartatthesametimepoint.After6dailyrepeatedimmobilizationstresses,thestressedratshada

    significant(P<0.05,VSthatofday0)lossofbodyweight(90%ofcontro1)andtheykeptlosingweightduringthewholeobserving

    time.Datawerepresentedasmean_+S.E.M.throughoutthepaper.

    1000

    750

    So0

    250

1o0

    Marlce~COntrolBDNFDR

    Fig.2RT-PCRidentificationofBDNF,KDRandBaxmRNAsinthenormallivertissue Notes:RT-PCRamplificationoflivertissueidentifiedPeRproductscorrespondingtOtheexpectedsizeforBDNF,KDRand

    BaxmRNAs,respectively.Marker:DL2000DNAmarkeridentifiedDNAproductsinsizeofbp;Control:Templatewasomittedto

    checkthestringencyofRTPeRsystem.

    2.AcutebutnotRepeatedImmobilizationIncreasedmRNALevelofKDR

    RT-PCRfromnormallivertissueidentifiedDNAproductsforBDNF,KDRandBaxattheirexpectedsize,

    respectively(Fig.2).

    KDR,asawellacceptedreceptorinmediatingVEGF'Sangiogenesisfunction,isexpressedonactivated

    HSCsandhepaticsinusoidalendothelialcells(SECs)

    ,J.OurRT-PCRexperimentrevealedanabundantexpression

    ofKDRmRNAinthecontrolratliver.Afteroneacuteimmobilization,itslevelincreasedsignificantly(P=O.005

    comparedwiththatofcontro1)to114.8%ofcontrolleve1.After15repeateddailyimmobilizations,themRNA

    levelforKDRincreasedslightlywithoutanysignificantdifference(Fig.3). 13

    一一o.Icouoo/0c0o?

    DifferentRolesofBDNF.KDRandBaxinLiverResponsetoAcuteorRepeatedImmobilizationStress:

    EvidencefromQuantifiedReverseTranscription-polymeraseChainReactionResearch }(DR

    G_aetin

    ControlAcutestressRepeatedstress

    Fig.3RepresentativeRT?PCRandhistogramsummaryresults(n=4pergroup)fromRT-PCRanalysisofmRNAlevelfor

    KDRinlivertissuetakenfromcontrolrats,ratsunderacuteorrepeatedimmobilizationstress Notes:Top:SampleRT-PCRshowingexpressionofKDRmRNAintheliver.Bottom:RelativedensityofKDRmRNA

    normalizedtOthatofinternalcontrol

    actin.KDRmRNAlevelsignificantlyincreasedafteranacuteimmobilizationstressrP=0.005

    VScontro1).After15repeateddailyimmobilizationstresses,themRNAlevelofliverKDRincreasedslightly.

    3.RepeatedbutnotAcuteImmobilizationDecreasedmRNALeveIofBDNF

    BDNFwasfoundtoexpresswithinthelivertissueofthecentrelanimalswhichsuggestedalocalsynthesis

    OfBDNFinlivertissue.ThelocaloriginationofBDNFiSHSCs".ItsmRNAlevelslightlyi

    ncreasedbutwithout

    significantdifierencefromcentrellevelafterasingleacuteimmobilizationstress.Afterl5repeateddaily

    immobilizationstresses.themRNAlevelofliverBDNFdecreasedsignificantly(P<0.0lcomparedwiththatof

    contro1)toaround82.22%ofcontrollevel(Fig.4).

    8DNF

    act

    ControlAcutestressRepeatedsb'ess

    Fig.4RepresentativeRT?PCRandhistogramsummaryresults(n--4pergroup)fromRT?PCRanalysisofmRNAlevelfor

    BDNFinlivertissuetakenfromcontrolrats.i'atsunderacuteorrepeatedimmobilizationstress

    Notes:ToD:SampleRT-PCRshowingexpressionofBDNFmRNAintheliver.Bottom:RelativedensityofBDNFmRNA

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