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1、无血清体外培养树突状细胞的研究作者：张怀东，宋振岚,李伟平【摘要】本研究的目的是建立对外周血来源的树突状细胞(dendriticcell,DC)的无血清培养方案。以含胎牛血清(FCS)、人AB血清的培养液作为对照。分离健康志愿者外周血单个核细胞，在不同培养液中分别加入GM-CSF、IL-4培养6天，再分别加入钙离子载体A23187继续培养24小时，于倒置显微镜下观察细胞形态，流式细胞术分析细胞表型，MTT比色法检测各组DC刺激同种异体外周血T细胞增殖的能力和DC刺激的T细胞对K562细胞的杀伤作用
2、。结果表明：无血清培养组培养出典型形态的DC，其表面CD14分子的表达明显减少，CD83、HLA-DR、CDw123分子的表达明显增高，具有明显的刺激同种异体T细胞增殖的能力和DC刺激的T细胞对K562细胞的杀伤作用。无血清组与两个含血清的对照组比较，组间无显著性差异。结论：无血清培养液培养的DC与含血清的培养液培养的DC相比，无显著性差异。DC无血清培养具有临床应用前景。【关键词】树突状细胞；无血清培养液；钙离子载体；外周血InVitroCultivationofDendriticCellswi
3、thSerum-freeMediumAbstractThisstudywasaimedtoinvestigatetheprotocolinvitrotoincubatethedendriticcell(DC)derivedfromperipheralbloodmonocytesusingserum-freemediumX-VIVO0.Peripheralbloodmonocytesfromhealthydonorsweretreatedwith100ng/mlGM-CSFand00U/mlIL-4
4、,respectively.Aftercultivationfordays,theyweretreatedwith100ng/mlcalciumionophoreA23187.Aftercultivationforhoursthecellularmorphologywasobservedunderinvertmicroscope,thesurfacemarkerswereanalyzedbyflowcytometry,theproliferationofallogeneticTcellswasde
5、tectedbyMTTcolorimetry,thespecificcytotoxicityofTcellsprimedwithDCwasexaminedbyMTTassay.Theresultsshowedthatinallthreegroupswithserum-free,fetalcalfserum(FCS)andhumanABserummediums,cellsdisplayedcharacteristicmorphologicalfeaturesofDC.SimultaneouslyCD
6、1expressionwasdecreased,andCD83,HLA-DRandCDw12expressionwereincreasedonthesecells.Inaddition,DCsculturedwiththesemethodscouldevidentlystimulatetheproliferationofallogeneticTcell.Ascomparedwiththetwocontrolsofserumcontaininggroups,theculturedcellsinthe
7、serum-freegroupsshowedalmostthesameallo-stimulatorycapabilityandcellularmorphologyandsurfacemarkers,andTlymphocytesprimedwiththeculture-derivedDCexhibitedthesimilarkillingactivitytoK56(P>).ItisconcludedthatthereisnosignificanceinDCnumbers,morphology,e
8、pitopeandabilitytostimulatetheproliferationofallogeneticTcellsbetweenDCinducedbyserum-freeX-VIVO0mediumandDCinducedbyserum-containedmedium.DCculturedandinducedbyserum-freemediumisworthusinginpracticewidely.Keywordsdendriticcell;serum-freemediu
