重组人可溶性CD40配体对树突状细胞体外分化、成熟及功能的影响

用Ficoll密度离心及贴壁法获得外周血单个核细胞 (PBMC ) ,PBMC经细胞因子组合诱导分化成树突状细胞 (DC ) :GM CSF (10 0ng/ml)与IL 4 (5 0ng/ml)诱导 5d后 ,分别加入TNF α (10ng/ml)或rhsCD4 0L (2 μg/ml)继续培养 4d ;倒置显微镜下观察DC形态 ,免疫荧光标记和流式细胞术分析DC表型 (CD1a、CD80、CD83、HLA DR、CD14、CD16、CD19)及摄取FITC Dextran抗原的能力 ;3 H TdR掺入法检测DC刺激自体混合淋巴细胞体外增殖反应 (MLR )能力 ;ELISA法分析DC培养上清中IL 12的水平 ;Trans well细胞趋化实验检测DC对自体外周T淋巴细胞的趋化能力。发现经rhsCD4 0L刺激的DC表面分子 (CD1a、CD80、CD83、HLA DR )的表达水平高于经典的细胞因子组合组 (GM CSF +IL 4 +TNF α ) ,同时rhsCD4 0L刺激后的DC摄取FITC Dextran的能力下降而刺激自体MLR和分泌IL 12的能力明显提高 ;而且rhsCD4 0L诱导的DC表面趋化因子受体CXCR4的表达水平及对自体外周T淋巴细胞的趋化能力均强于TNF α或FL激发的DC。rhsCD4 0L在体外不仅具有显著的诱导DC分化 ,促进DC成熟的功能 ,而且经rhsCD4 0L作用的DC能更有效地激发T淋巴细胞

Recombinant Human Soluble CD40 Ligand Exerts Powerful Lmpacts on the Devel-opment and Functions of Monocyte-derived DC in Vitro

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation of heparinized blood,and PBMC were plated into 6 well plates for 3 hour incubation The non adherent cells were removed and the adherent cells (monocytes) were cultured with GM CSF (100 ng/ml) and IL 4 (50 ng/ml) for 5 days Then TNF α (10 ng/ml) or recombinant human soluble CD40 ligand (rhsCD40L) (2 μg/ml) was respectively added to the cultures for another 4 days' induction Cell morphology was monitored by light microscopy and cell surface molecules including CD1α,CD80,CD83,and HLA DR by flow cytometry The induced DC functions were estimated by detecting the capacities of DC up take antigens with FITC Dextran capturing assay,of stimulating autogenetic lymphocyte in vitro proliferation with 3 H TdR insertion method,of chemotaxying T lymphocyte with Trans well and of secreting IL 12 with ELISA It was found that rhsCD40L (2 μg/ml) successfully developed peripheral blood monocytes into mature DC combined with GM CSF and IL 4,which with the typical morphology,special surface markers like CD1α,CD80,CD83,and HLA DR,with the full capacities of uptaking FITC Dextran particle,stimulating autogenic lymphocytes to proliferation,driving T lymphocytes to migration and IL 12 secretion Moreover,our results demonstrated that the expression levels of some immune co stimulators on DC including CD80 and HLA DR were higher than those DC induced with GM CSF+IL 4+TNF α Also the rhsCD40L derived mature DC expressed higher levels of CXCR4 and attracted more number of T lymphocytes than DC induced with TNF α or FL These results suggest that rhsCD40L has the potential role in stimulating the differentiation and maturation of monocyte derived DC and may be a more efficient stimulator for T lymphocytes