G-CSF动员的供体外周血树突状细胞免疫生物学特性的研究

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。

Immunobiologic properties of dendritic cells derived from G-CSF mobilized peripheral blood

Objective To explore the possibility of inducing DCs derived from donors′ PBMCs mobilized by G-CSF and collected with CS-3000plus,and to analyze their immunobiologic properties.Methods The adherent monocytes were propagated in serum-free medium combined with GM-CSF and IL-4 for 7 days and then divided into four groups: no stimulation group,TNF-α group,IL-10 plus TNF-α group and ATG plus TNF-α group.The peripheral blood of healthy blood donors served as control.There were five samples in each group,and each sample was tested in double.Cells were induced with IL-10 or ATG for one day,and then induced with TNF-α for another day.The immunophenotype,secretion of IL-12(P70),antigen phagocytosis and allogeneic stimulation response were examined.Results Immature DCs after 9-day induction were obtained.The expression of HLA-DR,CD11c,CD40,CD80,secretion of IL-12,and the activity of allogeneic stimulation response were all increased after induction with TNF-α.IL-10 increased the expression of CD1a.Both IL-10 and ATG inhibited the expression of HLA-DR,CD11c,CD40 and CD80.Compared to mature DCs,IL-12 secretion and activity of allogeneic stimulus response of DCs induced by IL-10 group and ATG group were decreased,while the function of antigen uptake was enhanced.However,the biological properties of DCs induced by IL-10 and ATG were different from each other.Compared with the DCs derived from the controls,the yield of DC,the expression of HLA-DR and CD11c,the expression of CD11c,CD40 and CD80 after induction,the secretion of IL-12,and the activity of allogeneic stimulus response all were decreased in DCs derived from G-CSF mobilized donors.Conclusions Immature DCs could be generated from monocytes via routine method.The mature DCs could be induced by TNF-α,and then acquire the tolerogenic properties with IL-10 or ATG.The immunophenotype and function of DCs from G-CSF mobilized donors were inferior to those from healthy blood donors.Although the yield of DCs was lower than that of the blood donors′,yet a large amount of PBMCs from a single donor could be obtained.Therefore the G-CSF mobilized PBMCs could probably become an important resource of different DCs.