摘要
目的探讨白细胞介素（IL）-10诱导的尘螨变应性哮喘患者外周血树突细胞（DC）对T淋巴细胞体外增殖的作用。方法选取2011年1—6月苏州大学附属第三人民医院呼吸科门诊收治的10例尘螨变应性哮喘患者外周血，用淋巴细胞分离液Fic011密度离心及贴壁法获得外周血单核细胞，加入人粒细胞巨噬细胞集落刺激因子（GM-CSF）＋IL4＋肿瘤坏死因子（TNF）-α常规诱导培养，同时加或不加细胞因子IL-10，经尘螨变应原负载后，运用免疫荧光标记和流式细胞术检测IL-10诱导的DC（DC-10）和无IL-10诱导的DC（DC-TNF）表面共刺激分子CD80、CD83、CD86、人白细胞抗原-DR（HLA—DR）和免疫球蛋白样转录体2（ILT2）的表达以及DC体外摄取异硫氰酸荧光素（FITC）-葡聚糖抗原的能力；将DC-10（DC．10刺激组）、DC．TNF（DC．TNF刺激组）或DC．10＋DC-TNF（联合刺激组）按一定比例与自体T淋巴细胞共培养，噻唑蓝（MTT）法检测T淋巴细胞体外增殖；酶联免疫吸附试验（ELISA）法检测各组T淋巴细胞分泌细胞因子1干扰素（IFN-r）、IL-4、IL-5和IL．13的水平。结果与DC-TNF相比：抗原负载的DC-10表面标志成熟的共刺激分子CD80、CD83、CD86和HLA．DR的表达显著下调；ILT2的表达上调；摄取FITC-葡聚糖的能力增强（72．32％±2．93％比54．41％±2．95％，P〈0．01）。与DC-TNF刺激组（1．74±0．15）相比，DC-10刺激组（1．064-0．18）和联合刺激组（1．34±0．16）的T淋巴细胞增殖显著减弱（均P〈0．01）。DC-10刺激组T淋巴细胞分泌的IFN一1、IL4、IL-5、IL-13分别为（29984-141）、（1574-17）、（26084-254）、（554-11）w,／L，联合刺激组T淋巴细胞分泌的IFN-r、IL4、IL-5、IL·13分别为（3223±203）、（1494-19）、（24654-183）、（88±10）ng／L，均显著低于DC-TNF刺激组的（36394-209）、（173±16）、（2771±183）、（1274-11）ng／L（均P〈0．01）。结论IL-10诱导的DC具有耐受型DC的表型及功能，能有效减弱变应性T淋巴细胞反应。
Objective To explore the roles of interleukin （IL）-10 differentiated peripheral blood monocyte-derived dendritic cell （DC-10） of allergic asthma patients in T-lymphoeytes proliferation in vitro. Methods From January to June 2011, 10 subjects with dust mite allergic asthma treated at Third Affiliated Hospital of Soochow University were enrolled. Their peripheral blood monocytes were isolated by Fieoll- Hypaque solution density gradient centrifugation and adherent method. And the adherent monocytes were routinely cultured with granuloeyte-macrophage colony-stimulating factor （GM-CSF） ＋ interleukin-4 （IL-4） ＋ tumor necrosis factor-alpha （ TNF-α）, stimulated with/without interleukin-10 （ IL-10 ）, pulsed with dust mite allergen and finally harvested. The cell surface molecules including CD80, CD83, CD86, human leukocyte antigen （HLA）-DR and immunoglobulin-like transcript 2 （ILT2） were detected by immunofluorescent labeling and flow cytometry. And cellular functions were estimated by detecting the capacities of DC uptake antigens with fluoreseein isothiocyanate （FITC）-dextran capturing assay. The IL-10differentiation DC （DC-10） were cultured with autologous peripheral T cells （DC-10 group）, either alone （DC-TNF group） or together （combined group） with autologous immunostimulatory DC （DC-TNF）. And the impact of this treatment on T-cell responses was assessed for each donor by 3-（4, 5 ）-dimethylthiahiazo （ -z-y1 ） -3, 5-di-pbenytetrazoliumromide （MTT） assay. The production of interferon-y,/ （ IFN-y, ）, IL-4, interlenkin-5 （IL-5） and intedeukin-13 （IL-13） were measured with the quantification of enzyme linked immunosorbent assay （ELISA） kits. Results In DC-10, the levels of some mature DC＇s markers （CD80, CD83, CD86 ＆ HLA-DR） decreased, ILT2 increased and there were the higher capacities of up-taking FITC-dextran particle （72. 32% ±2. 93% vs 54. 41% ± 2. 95% , P 〈 0. 01 ）. Compared with the DC-TNF group （1.74 ±0. 15） , the T cell proliferation of the DC-10 group （1.06±0. 18） and that of the combined group （1.34 ± 0. 16） were significantly inhibited （P 〈 0. 01 ）. The secretion levels of IFN-y, IL-4, IL-5 and IL-10 were （2998 ±141）, （157±17）, （2608±254） and （55 ±11） ng/L in the DC-10 group versus （3223 ± 203 ）, （ 149 ± 19 ）, （2465 ± 183 ） and （ 88 ±10 ） ng/L respectively in the combined group. They were all significantly reduced versus （3639 ±209） , （173 ± 16） , （2771 ±183 ） and （127 ± 11 ） ng/L in the DC-TNF group （ all P 〈 0.01 ）. Conclusions The IL-10-treated human DC may express a tolerogenic phenotype and induce the allergen tolerance of T cells. And the induction of DC-10 represents a promising target for therapeutic intervention of effectively managing the clinical outcomes of allergic asthma.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2012年第40期2851-2854,共4页
National Medical Journal of China
