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目的探讨从以G-CSF动员、CS-3000 plus血细胞分离机采集的供者外周血中诱导产生不同特性树突状细胞(DC)的可行性及DC的生物学特性。方法实验组为用CS-3000 plus血细胞分离机采集的5名造血干细胞捐献者的单核细胞(PBMCs),PBMCs培养贴壁后,经含IL-4、GM-CSF的无血清培养基诱导培养7 d,进一步分为4组:未刺激、肿瘤坏死因子α(TNF-α)、IL-10+TNF-α、抗胸腺球蛋白(ATG)+TNF-α组;对照组为5名健康献血者周血,亦按上述条件分组,每组复孔。后2组分别为IL-10、ATG诱导1 d后再加入TNF-α诱导1 d;分别测定各组DC的免疫表型、IL-12(P70)分泌、抗原吞噬以及异基因T淋巴细胞刺激反应。结果诱导培养9 d后可获得非成熟DC(iDC),单纯TNF-α刺激后,HLA-DR、CD11c、CD40、CD80的表达均明显增高,IL-12分泌增加,对异基因T淋巴细胞刺激反应性增强;而IL-10可以使CD1a表达上调,IL-10和ATG均抑制HLA-DR、CD11c、CD40、CD80的表达;与诱导的成熟DC相比,IL-10、ATG诱导DC的IL-12(P70)的分泌、对异基因淋巴细胞刺激反应性均减低,抗原摄取能力增强,但ATG、IL-10诱导的DC生物学特性不完全一致;动员的供体与对照健康献血者诱导的DC在产率,HLA-DR、CD11c表达,成熟诱导后CD11c、CD40、CD80表达,IL-12分泌,异基因T细胞刺激反应性等相比均明显减低。结论G-CSF动员的供体单核细胞PBMCs通过常规诱导可以产生iDC,TNF-α可诱导其分化成熟,IL-10、ATG可以使其获得致耐受的特性;与健康献血者相比,G-CSF动员的供体DC表型和功能部分减低,;虽然动员的供体DC产率不及健康献血者,但血细胞分离机可以可采集到大量的单个核细胞(PBMCs),因此G-CSF动员的外周血有望成为不同DC的重要来源途径。
Objective To investigate the feasibility of dendritic cells (DCs) induced by G-CSF mobilization and peripheral blood collected from CS-3000 plus hemocyte separator and the biological characteristics of DCs. Methods The experimental group consisted of 5 mononuclear cells (PBMCs) from 5 hematopoietic stem cell donors collected by CS-3000 plus blood cell separator. PBMCs were cultured in vitro and cultured in serum-free medium containing IL-4 and GM-CSF 7 d, and further divided into 4 groups: unstimulated, TNF-α, IL-10 + TNF-α and ATG + TNF- Week of blood, also grouped according to the above conditions, each hole complex. IL-10 was induced in the latter two groups, and induced by TNF-α for 1 d after ATG induction for 1 day. Immunophenotype, secretion of IL-12 (P70), phagocytosis of antigen and allogeneic T lymphocyte stimulated reaction. Results After cultured for 9 days, immature DCs (iDC) were obtained. The expressions of HLA-DR, CD11c, CD40 and CD80 were significantly increased and the secretion of IL-12 increased after stimulation with TNF- IL-10 and ATG both inhibited the expression of HLA-DR, CD11c, CD40 and CD80; compared with the induced mature DC, IL-10 and ATG induced DC IL -12 (P70) secreted decreased the allogeneic lymphocyte stimulation reactivity and enhanced the antigen uptake capacity, but the biological characteristics of DC induced by ATG and IL-10 were not exactly the same; the mobilized donor and the control healthy donor induced Of DCs were significantly lower than that of HLA-DR, CD11c, CD11c, CD40, CD80, IL-12 secretion and allogeneic T cell stimulatory reactivity. Conclusion G-CSF-mobilized donor mononuclear cells (PBMCs) can induce iDC by conventional induction. TNF-α induces differentiation and maturation. IL-10 and ATG can induce tolerance of PBMCs. Compared with healthy donors, G-CSF-mobilized donor DC phenotypes and functional portions are reduced; while mobilized donor DCs yield less well than healthy blood donors, haemocyte separators can harvest large numbers of mononuclear cells (PBMCs) and thus G-CSF Mobilized peripheral blood is expected to be an important source of different DCs.