维生素A对脐血树突状细胞分化与成熟及其功能的影响

Effects of vitamin A on the differentiation, maturation and functions of dendritic cells from cord blood.

目的 研究维生素A在体内的代谢活性产物视黄酸 (retinoicacid ,RA)对树突状细胞(dendriticcells ,DC)分化、成熟及功能的影响 ,进一步探索维生素A对免疫功能调节的机理。方法 取健康足月儿脐血 9份分离单个核细胞后 ,进行体外培养诱导DC的同时加入生理浓度的RA ,采用流式细胞仪检测DC的表面分子CD1a、CD83、HLR DR以判断RA对DC分化、成熟的影响 ;混合淋巴反应的强度观测RA对DC抗原递呈功能的作用 ;荧光定量PCR法检测细胞因子IL 12p35、IL 12p4 0、IFN γ(Th1)、IL 4、IL 10 (Th2 )mRNA转录水平 ,分析RA对DC诱导Th细胞分化的影响。结果 培养第 6天RA使DC数量显著下降 (P <0 0 0 1) ,培养第 9天 ,DC总数相仿 ,但RA处理后不成熟DC百分比增高 ,而成熟DC百分比降低 ,差异有显著意义 (P <0 0 0 1) ;DC混合淋巴反应的cpm值降低2 9 4 % (P <0 0 0 1)。细胞因子IL 12、IFN γmRNA下降 ,而IL 4、IL 10的mRNA水平升高 ,差异均具有显著意义。结论 维生素A延迟体外培养的脐血单个核细胞向DC的分化和成熟 ,且降低其混合淋巴细胞反应的能力 ,减少Th1细胞因子的产生而增加Th2细胞因子的产生 ,使免疫反应向Th2方向偏移。

Objective It is well known that vitamin A can improve mucosal immunity and anti-infection immunity. But the mechanisms thereof remain to be clarified. Previous studies on the role of vitamin A in immune regulation focused on lymphocytes, whereas little had been done about dendritic cells, which play very important roles in immune response. The objective of this study was to understand the effects of retinoic acid (RA), the metabolic product of vitamin A in vivo,on the differentiation, maturation and functions of dendritic cells from cord blood.Methods Cord blood samples were collected from nine well-nourished full-term neonates. Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and cultured in the presence of 1 000 u/ml GM-CSF, 500 u/ml IL-4 for 6 days, then TNF-α 20 ng/ml was added into the medium and cultured for another 3 days. The cells were incubated with or without 1×10 -6MRA. Expression of surface molecules, CD1a, CD83, HLA-DR on DC was measured by flow cytometry. The ability of DC derived from the culture to induce proliferation of T cells in the mixed lymphocyte reaction (allo-MLR) was used for the evaluation of their function. IL-12, IFN-γ, IL-4 and IL-10 were detected at mRNA levels by RT-PCR to understand the roles of DC treated with RA in regulation of Th1/Th2 balance. Results On the sixth day of cell culture, the percentage of DC incubated with RA(57.28±9.22) was much lower than that without RA (79.57±11.85) (P<0.001), but on the ninth day, there were no differences between the presence or absence of RA(76.18±10.27 vs. 73.72±15.58). When RA was added to the medium and the culture was continued for nine days, the percent of immature DC (CD1a+HLA-DR+) was much higher than that of the control (absence of RA)(58.93±4.70 vs. 45.80±7.88, t=6.575 , P<0.001); whereas, mature DC (CD83+HLA-DR+) percentage was markedly lower than that of the control (17.25±8.49 vs. 27.92±13.94 , t=4.435, P=0.002). The T lymphocytes proliferation induced by the DC treated with RA was reduced from 16 857±3 643 to 11 924±2 576 cpm (t=5.598, P<0.001) in allo-MLR. Expression of mRNA for IL-12p35, IL-12p40, IFN-γ in the cells that had been incubated with RA declined, but IL-10, IL-4 increased signifirantly. Conclusion Vitamin A inhibited the differentiation and maturation of cord blood DC, reduced it's ability to stimulate allo-T lymphocytes proliferation, and down-regulated Th1 cytokines, up-regulatedTh2 cytokines, consequently made immune response inclined to Th2.