小鼠脾脏来源树突状细胞的体外扩增培养【精品论文】.doc

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1、小鼠脾脏来源树突状细胞的体外扩增培养作者：小小小作者：黎辉，曹宇皎，黄军华，刘俊峰【摘要】n的：对体外诱导小鼠脾脏单个核细胞分化成的树突状细胞(do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的白细胞介索4仃L4)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：小鼠脾脏來源的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

2、达CDllc、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乂一可靠来源。【关键词】脾脏；骨髓；树突状细胞；诱导；小鼠Dendriticcells(DC)arethemostpotentantigenpresentingcellsthatareresponsibleforprimingnativeTcel1sintheimmuneresponse-I

3、mmunotherapyofDC/tumorvaccinehasbecomeanimportanttherapeuticstrategyagainsttumors[12]•However,thebasicandclinicalapplicationsofDCshavebeenlimitedbythelowyieldandpurityofDCsgeneratedbytraditionalbonemarrowderivation.Inthisstudy,weinducedmousespleenmononuclearcellsi

4、ntodendriticcellsinvitro,andthencomparedthebiologicalcharacteristicsofthemwiththoseofbonemarrowmononuclearcellsderivedDCs.1Materialsandmethods1.1MaterialsMainreagents:recombinantmousegranulocytemacrophagecolonystimulatingfactor(GMCSF),recombinantmouseinterleukin4(

5、IL4),recombinantmouseleukemiainhibitoryfactor(LIF,Peprotech),lipopolysaccharide(LPS,Sigma);mitomycinC(MMC)andmethabenzthiazuron(MTT,Duchfo).DCsculturemedium:highglucoseDMEMplus15%fetalcalfserumplus25mg•L~1rmGMCSFandrmlL4.Mice:sixtoeightweeksoldfemaleBALB/cand129mi

6、cewerepurchasedfromAnimalCenterofGuiyangMedicalCol1ege.2MethodsInductionofDCfrommousespleenCellswereflushedoutofspleenof129mice.Mononuclearcellsattheinterfacewerecollectedbycentrifugationoveralympholytegradient.Theywerewashedandthenculturedin6wellplatefor2hwiththe

7、densityof5X104mileThesuspendedcellswereremovedandtheadherentcellswereculturedwithDCculturemedium.LPSwasaddedonday7andthesuspendedcel1swereharvested24hlater,whi1eatthesametimemousebonemononuclearcellswereinducedintoDCs.MorphologyobservationCellswereobservedundermi

8、croscopedailyandsuspendedcellswereidentifiedbyelectronmicroscopy.2.3CellsurfacemarkeranalysisCellswerecollectedandaddedintocentrifugetube.MixedwithFITCC