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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（PBMNC），在不同培养液中分别加入GM-CSF（100ng/ml）、IL-4（500U/ml）培养6天，再分别加入钙离子载体A23187（100ng/ml）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K
2、562细胞的杀伤作用。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（P>0.05）。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】16树突状细胞；无血清培养液；钙离子载体；外周血 InVitroCultivationofDendriticCellswit
3、hSerum-freeMedium AbstractThisstudywasaimedtoinvestigatetheprotocolinvitrotoincubatethedendriticcell(DC)derivedfromperipheralbloodmonocytesusingserum-freemediumX-VIVO20.Peripheralbloodmonocytesfromhealthydonorsweretreatedwith100ng/mlGM-CSFand500U/mlIL-4,respectively.Aftercul
4、tivationfor6days,theyweretreatedwith100ng/mlcalciumionophoreA23187.Aftercultivationfor24hoursthecellularmorphologywasobservedunderinvertmicroscope,thesurfacemarkerswereanalyzedbyflowcytometry,theproliferationofallogeneticTcellswasdetectedbyMTTcolorimetry,thespecificcytotoxicit
5、yofTcellsprimedwithDCwasexaminedbyMTTassay.Theresultsshowedthatinallthreegroupswithserum-free,fetalcalfserum(FCS)andhumanABserummediums,cellsdisplayedcharacteristicmorphologicalfeaturesofDC.SimultaneouslyCD14expressionwasdecreased,andCD83,HLA-DRandCDw123expressionwereincreased
6、onthesecells.Inaddition,DCsculturedwiththesemethodscouldevidentlystimulate16theproliferationofallogeneticTcell.Ascomparedwiththetwocontrolsofserumcontaininggroups,theculturedcellsintheserum-freegroupsshowedalmostthesameallo-stimulatorycapabilityandcellularmorphologyandsurfacem
7、arkers,andTlymphocytesprimedwiththeculture-derivedDCexhibitedthesimilarkillingactivitytoK562(P>0.05).ItisconcludedthatthereisnosignificanceinDCnumbers,morphology,epitopeandabilitytostimulatetheproliferationofallogeneticTcellsbetweenDCinducedbyserum-freeX-VIVO20mediumandDCin
8、ducedbyserum-containedmedium.DCculturedandinducedbyserum-free
