浅论小鼠脾脏来源树突状细胞的体外扩增培养.docx

浅论小鼠脾脏来源树突状细胞的体外扩增培养.docx

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浅论小鼠脾脏来源树突状细胞的体外扩增培养

浅论小鼠脾脏来源树突状细胞的体外扩增培养

【摘要】目的：

对体外诱导小鼠脾脏单个核细胞分化成的树突状细胞（DC）的生物学表型及功能进行检测，并与骨髓来源的DC进行比较。

方法：

分离小鼠脾脏单个核细胞，在体外培养的条件下通过添加25ng·ml-1的粒单核细胞集落刺激因子和25ng·ml-1的白细胞介素4（IL4）将其诱导为DC，分别从细胞形态、表型以及功能3个方面对其进行检测，并与骨髓来源的DC进行比较。

结果：

小鼠脾脏来源的DC经磷酸脂多糖（LPS）刺激成熟后其表面显示出明显的树枝状突起，高表达CD11c、CD86及MHCⅡ类分子，并具有极强的刺激同种异基因淋巴细胞增殖的能力，其特征与骨髓来源的DC相差无几。

结论：

小鼠脾脏单个核细胞能够被诱导为具有正常形态、表型、功能的DC，为DC的功能研究以及进一步制备相应的肿瘤疫苗提供又一可靠来源。

【关键词】脾脏;骨髓;树突状细胞;诱导;小鼠

　Dendriticcells（DC）arethemostpotentantigenpresentingcellsthatareresponsibleforprimingnativeTcellsintheimmuneresponse.ImmunotherapyofDC/tumorvaccinehasbecomeanimportanttherapeuticstrategyagainsttumors[12].However,thebasicandclinicalapplicationsofDCshavebeenlimitedbythelowyieldandpurityofDCsgeneratedbytraditionalbonemarrowderivation.Inthisstudy,weinducedmousespleenmononuclearcellsintodendriticcellsinvitro,andthencomparedthebiologicalcharacteristicsofthemwiththoseofbonemarrowmononuclearcellsderivedDCs.

　　1Materialsandmethods

　　Materials

　　Mainreagents:

recombinantmousegranulocytemacrophagecolonystimulatingfactor（GMCSF）,recombinantmouseinterleukin4（IL4）,recombinantmouseleukemiainhibitoryfactor（LIF,Peprotech）,lipopolysaccharide（LPS,Sigma）;mitomycinC（MMC）andmethabenzthiazuron（MTT,Duchfo）.DCsculturemedium:

highglucoseDMEMplus15%fetalcalfserumplus25mg·L-1rmGMCSFandrmIL4.Mice:

sixtoeightweeksoldfemaleBALB/cand129micewerepurchasedfromAnimalCenterofGuiyangMedicalCollege.

　　Methods

　　InductionofDCfrommousespleenCellswereflushedoutofspleenof129　cellsattheinterfacewerecollectedbycentrifugationoveralympholytegradient.Theywerewashedandthenculturedin6wellplatefor2hwiththedensityof5×104　suspendedcellswereremovedandtheadherentcellswereculturedwithDCculturemedium.LPSwasaddedonday7andthesuspendedcellswereharvested24hlater,whileatthesametimemousebonemononuclearcellswereinducedintoDCs.

　　MorphologyobservationCellswereobservedundermicroscopedailyandsuspendedcellswereidentifiedbyelectronmicroscopy.

　　CellsurfacemarkeranalysisCellswerecollectedandaddedintocentrifugetube.MixedwithFITCCD11candMHCⅡPE,FITCCD86andMHCIIPE,respectively,thecellswerekeptat4℃for30min.AfterbeingwashedtwicebyPBS,cellswereassayedbyflowcytometry.

　　Mixedlymphocytereactions（MLR）CollectedspleenderivedDCwereaddedwith25μg·ml-1MMCasastimulatorfor45minandwashedtwice.NylonwoolpurifiedTcel1sasresponderswereaddedin96wellroundbottomplatesatvariousratiosfor4　thesametime,wesetcontrolgroupthatdidntincludestimulators.4hbeforeharvesting,cellswerepulsedwith20μlMTT（5mg·ml-1）,thenaddedwith100μlDMSOanditsabsorbencywasmeasured.Thestimulationindexwascalculatedaccordingtothefollowingformula:

SI=absorbencyofstimulationgroupabsorbencyofcontrast/theabsorbencyofcontrast.

　　Statisticalanalysis

　　Dataareexpressedas±s　resultsoftheanalysiswasshownbyt　valueof　wasconsideredstatisticallysignificant.

　　2Results

　　MorphologicalcharacteristicsofmousespleenderivedDCs

　　Atthefirst2-4d,themousespleenmononuclearcellsgrewslowlyandroundinshape,thebulkwassmall,thepopulationexpandedrapidlywithtimeandthenthebulkbecamelarger　spleenderivedDCsalreadyhadirregulardendritesafterbeingculturedfor5-7d.Afteradding1μg·ml-1ofLPSfor24h,thedramaticveilsofcytoplasmandextensivedendritesweremoredistinct（Fig1）,justasthematureBMDCs.Besides,5-10cellconglomeratescouldbeseen（Fig2）.Byelectromicroscopythedramaticveilsofcytoplasmandextensivedendriteswereseenmoreclearly（Fig3）.

　　Analysisofsurfacephenotype

　　DCsderivedfrommousespleenmononuclearcellswereinducedtomaturethroughexposuretoLPSandhighexpressionofsurfacemoleculesMHCⅡandCD86wasassociatedwithantigen　expressionofpeculiarsurfacemoleculeCD11c（Fig4）inspleenDCswasjustasthatinBMDCs.

　　MLR

　　FollowingexposuretoLPS,mousespleenderivedDCswereinducedandbecamemature,whichdemonstratedtheircapacitytostimulatepotentprimaryTcellresponsesinmixedlymphocytecultures（Tab1）.Tab1StimulationindexforprimaryTcelloftwosourcederivedDCs（±s）

　　3Discussion

　　DCsareuniqueamongpopulationsofantigenpresentingcellsbythevirtueoftheircapacitytodirecttheoutcomeofantigenrecognitionbynaiveTcells.DCsintheperipherycaptureandprocessantigens,expresslymphocytecostimulatorymolecules,migratetolymphoidorgansandsecretecytokinestoinitiateimmune　studieshaveshownthatDCsplaycrucialrolesincontrollingtumorgrowth,graftrejectionand　present,wehavealreadyinducedDCsfrombonemarrow,etc.[57],andwillproduceantitumorvaccinebygenetransfection,tumorassociatedantigenimpactionandcellfusion[810].Theeffectsofresearchinexperimentalanimalwereexciting.Furthermore,DCimmunotherapyhasbeenintroducedintheclinic,andhasbeenprovedtobefeasible,nontoxicandeffectiveinsomecancerpatients,andparticularlysowhentheDCsarecompletelymatureandactivated[11].Theresultsofthefirstclinicaltrialhavebeenpublished,andunequivocallydemonstratedthatonlymatureDCsarecapableofinducingpotentantiKLHspecificTcellandBcellresponse[12].ThelifecycleofDCincludesseveralstagescharacterizedbydistinctfunctionsandmechanismsofregulation[13].TheDCsderivedfromdifferentsourcesoratdifferentlifestagesmayhavedifferentbiologicalcharacteristics[1418].

　　Inthepresentstudy,weinducedDCsdifferentiatedfrommousespleenmononuclear　resultsshowedthatitcouldbeinducedintoDCswithnormalsurfacephenotypeinvitrobyadditionalappropriatesignalsintheformofgrowthfactorsand　dramaticveilsofcytoplasmandextensivedendriteswereseenintheirsurfacewhentheywereinducedandmaturedbyaddingLPS,andtheyexpressedthemyeloidpeculiarsurfacemoleculeCD11c,MHCⅡandCD86whichwereassociatedwithantigenpresenting.ComparedwithBMDC,thesurfacemorphologicalcharacteristicsofthemweresimilar.TheyhavealreadyhadafewirregulardendritesbeforematuritybyplusLPS,andexpressedlowerlevelsofCD11c,MHCⅡ,CD86.InducedintomaturebyLPS,thecharacteristicsweremoreevident.MousespleenmononuclearcellderivedmatureDCsdemonstratestheircapacitytostimulatepotentprimaryTcellresponsesinmixedlymphocytereactions.TherewasnostatisticaldifferencecomparedwithBMDC.Fromthisresearch,wecanconcludethatmousespleenderivedDCshaveanalogicalbiologicalcharacteristicstothoseinBMDCwhichhasbeenappliedinbasicandclinicalresearch.ThereforeitprovidesareliablesourcefortheresearchofDCsandthepreparationofantitumorvaccineinthenextstep.

【参考文献】

　[1]FLEMINGJN,HOSTIKKASL,CHENEY,etal.Plasmacytoiddendriticcellsandinterferonlevelsareincreasedinlymphaticmalformations[J].OtolaryngolHeadNeckSurg,2008，139（5）:

671676.

　　ZOUGM,MARTINSONJ,HUWY,etal.TheeffectofLIGHTininducingmaturationofmonocytederiveddendriticcellsfromMDSpatients[J].CancerImmunolImmunother,2004,53（8）:

681689.

　　CHAUVINC,JOSIENR.Dendriticcellsaskillers:

mechanisticaspectsandpotentialroles[J].Immunol,2008,181:

1116.

　　BANCHEREAUJ,STEINMANRM.Dendriticcellsandthecontrolofimmunity[J].Nature,1998,392:

245252.

　　DOMINQUEZSOTOA,CORBIA　dendriticcelllectinsandtheirroleinimmuneresponses[J].CurrOpinInvestigDrugs,2007,8:

910920.

　　BRUNOL.Differentiationofdendriticcellsubsetsfrommousebonemarrow[J].MethodsMolBiol,2007,380:

4757.

　　OGAWAF,IINUMAH,OKINAGAK.Dendriticcellvaccinetherapybyimmunizationwithfusioncellsofinterleukin2genetransduced,spleenderiveddendriticcellsandtumourcells[J].ScandJImmunol,2004,59:

432439.

　　denBROKMH,NIERKENSS,FIGDORCG,etal.Dendriticcells:

toolsandtargetsforantitumorvaccination[J].ExpertRevVaccines,2005,4:

699710.

　　BANCHEREAUJ,PALUCKAA　cellsastherapeuticvaccinesagainstcancer[J].NatRevImmunol,2005,5:

296306.

　　[10]GONGJ,KOIDOS,CALDERWOODSK.Cellfusion:

fromhybridomatodendriticcellbasedvaccine[J].ExpertRevVaccines,2008,7:

10551068.

　　[11]AARNTZENEH,FIGDORCG,ADEMAGJ,etal.Dendriticcellvaccinationandimmunemonitoring[J].CancerImmunolImmunother,2008,57:

15591568.

　　[12]deVRIESIJ,LESTERHUISWJ,SCHARENBORGNM,etal.Maturationofdendriticcellsisaprerequisiteforinducingimmuneresponsesinadvancedmelanomapatients[J].ClinCancerRes,2003,9:

50915100.

　　[13]CHOWA,TOOMRED,GARRETTW,etal.DendriticcellmaturationtriggersretrogradeMHCclassⅡtransportfromlysosomestotheplasmamembrane

　　[J].Nature,2002,418:

988994.

　　[14]KATSNELSONA.Kickingoffadaptiveimmunity:

thediscoveryofdendriticcells[J].JExpMed,2006,203（7）:

1622.

　　[15]MENGESM,BAUMEISTERT,ROSSNERS,etal.IL4supportsthegenerationofadendriticcellsubsetfrommurinebonemarrowwithalteredendocytosiscapacity[J].JLeukocBiol,2005,77（4）:

535543.

　　[16]BASHYAMH.RalphSteinman:

dendriticcellsbringhometheLasker[J].JExpMed,2007,204（10）:

22452248.

　　[17]TANJK,ONEILLHC.MaturationrequirementsfordendriticcellsinTcellstimulationleadingtotoleranceversusimmunity[J].JLeukocBiol,2005,78

（2）:

319324.

　　[18]鲍文,陈宝安,丁家华.抗原递呈细胞在异基因造血干细胞移植中的研究进展[J].东南大学学报:

医学版,2006,25

（2）:

122125.