小鼠脾脏来源树突状细胞的体外扩增培养

《小鼠脾脏来源树突状细胞的体外扩增培养》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、小鼠脾脏来源树突状细胞的体外扩增培养作者：黎辉，曹宇皎，黄军华，刘俊峰【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MH

2、C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/tumorvacc

3、inehasbecomeanimportanttherapeuticstrategyagainsttumors[12].However,thebasicandclinicalapplicationsofDCshavebeen11limitedbythelowyieldandpurityofDCsgeneratedbytraditionalbonemarrowderivation.Inthisstudy,weinducedmousespleenmononuclearcellsintodendriticcellsinvitro,andthencomp

4、aredthebiologicalcharacteristicsofthemwiththoseofbonemarrowmononuclearcellsderivedDCs.　　1Materialsandmethods　　1.1Materials　　Mainreagents:recombinantmousegranulocytemacrophagecolonystimulatingfactor(GMCSF),recombinantmouseinterleukin4(IL4),recombinantmouseleukemiainhibitor

5、yfactor(LIF,Peprotech),lipopolysaccharide(LPS,Sigma);mitomycinC(MMC)andmethabenzthiazuron(MTT,Duchfo).DCsculturemedium:highglucoseDMEMplus15%fetalcalfserumplus25mg·L-1rmGMCSFandrmIL4.Mice:sixtoeightweeksoldfemaleBALB/cand129micewerepurchasedfromAnimalCenterofGuiyangMedicalCo

6、llege.　　1.2Methods11　　1.2.1InductionofDCfrommousespleenCellswereflushedoutofspleenof129mice.Mononuclearcellsattheinterfacewerecollectedbycentrifugationoveralympholytegradient.Theywerewashedandthenculturedin6wellplatefor2hwiththedensityof5×104ml-1.Thesuspendedcellswereremoveda

7、ndtheadherentcellswereculturedwithDCculturemedium.LPSwasaddedonday7andthesuspendedcellswereharvested24hlater,whileatthesametimemousebonemononuclearcellswereinducedintoDCs.　　1.2.2MorphologyobservationCellswereobservedundermicroscopedailyandsuspendedcellswereidentifiedbyelectron

8、microscopy.　　1.2.3CellsurfacemarkeranalysisCellswerecollected