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【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(IL4)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MHCⅡ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[12].However,thebasicandclinicalapplicationsofDCshavebeenlimitedbythelowyieldandpurityofDCsgeneratedbytraditionalbonemarrowderivation.Inthisstudy,weinducedmousespleenmononuclearcellsintodendriticcellsinvitro,andthencomparedthebiologicalcharacteristicsofthemwiththoseofbonemarrowmononuclearcellsderivedDCs.1MaterialsandmethodsMaterialsMainreagents:recombinantmousegranulocytemacrophagecolonystimulatingfactor(GMCSF),recombinantmouseinterleukin4(IL4),recombinantmouseleukemiainhibitoryfactor(LIF,Peprotech),lipopolysaccharide(LPS,Sigma);mitomycinC(MMC)andmethabenzthiazuron(MTT,Duchfo).DCsculturemedium:highglucoseDMEMplus15%fetalcalfserumplus25mg·L-1rmGMCSFandrmIL4.Mice:sixtoeightweeksoldfemaleBALB/cand129micewerepurchasedfromAnimalCenterofGuiyangMedicalCollege.MethodsInductionofDCfrommousespleenCellswereflushedoutofspleenof129cellsattheinterfacewerecollectedbycentrifugationoveralympholytegradient.Theywerewashedandthenculturedin6wellplatefor2hwiththedensityof5×104suspendedcellswereremovedandtheadherentcellswereculturedwithDCculturemedium.LPSwasaddedonday7andthesuspendedcellswereharvested24hlater,whileatthesametimemousebonemononuclearcellswereinducedintoDCs.MorphologyobservationCellswereobservedundermicroscopedailyandsuspendedcellswereidentifiedbyelectronmicroscopy.CellsurfacemarkeranalysisCellswerecollectedandaddedintocentrifugetube.MixedwithFITCCD11candMHCⅡPE,FITCCD86andMHCIIPE,r