小鼠不成熟CD8α＋树突状细胞的免疫抑制功能的体外实验  

The function of murine immature CD8a^＋ dendritic cells in vitro

作　　者：纳宁[1] 徐霖[1] 曹开源[1] 彭延文[1] 陈康[1] 项鹏[1] 李树浓[1] 

机构地区：[1]中山大学附属第三医院肾移植科,广州510630

出　　处：《中华器官移植杂志》2011年第1期6-10,共5页Chinese Journal of Organ Transplantation

摘　　要：目的 观察不成熟CD8α＋树突状细胞（DC）的体外免疫抑制功能.方法 取C57BL/6（H-2b）小鼠和Balb/c（H-2d）小鼠的骨髓和脾脏,制备不成熟CD8α＋DC和脾淋巴细胞,并经丝裂霉素处理.采用甲基噻唑基四唑（MTT）法,实验设混合淋巴细胞反应（MLR）组（阳性对照组）、MLR加不同密度的同系CD8α＋DC组、MLR加不同密度同种异型CD8α＋DC组、MLR加不同密度CD8α＋DC的培养上清组、CD8α＋DC加同系T淋巴细胞组及阴性对照组.MLR组按刺激细胞/反应细胞10∶1建立.CD8α＋DC按与反应细胞比例0.2∶1、0.5∶1、0.8∶1和1∶1的梯度加入MLR中分别建立同系CD8α＋D组和同种异型CD8α＋D组;MLR中加入不同密度（1×105/ml～5×106/ml）CD8α＋DC的培养上清液建立CD8α＋DC上清组;CD8α＋DC与同系淋巴细胞共培养建立CD8α＋DC加同系T淋巴细胞组;以反应细胞2×105/孔作为阴性对照.采用酶联免疫吸附试验法检测MLR加同系CD8α＋DC组（1∶1）上清液中γ干扰素（IF-γ）和白细胞介素10（IL-10）的浓度.结果 同系和同种异型不成熟CD8α＋DC对MLR均有抑制作用（P＜0.05）,二者间差异无统计学意义（P＞0.05）.抑制作用随DC比例的增加而增强,当CD8α＋DC/反应细胞比例大于0.2时显示抑制作用（P＜0.05）,比例为1时抑制作用最强.CD8α＋DC体外刺激同系淋巴细胞增殖的能力较弱,当DC与T淋巴细胞的比值大于2时,显示出一定的刺激作用（P＜0.05）,其培养上清液对MLR也有抑制作用（P＜0.05）,其中密度5×105/ml的细胞培养上清液抑制作用最强.MLR加同系CD8α＋DC组（1∶1）上清液中IL-10的含量为（451.9±12.2）pg/ml,IFN-γ的含量为（1.0±1.2）pg/ml.结论 不成熟CD8α＋DC体外具有免疫抑制或诱导免疫耐受的功能,可产生较高水平的IL-10,CD8α＋DC及其培养上清液均可抑制MLR.Objective To observe the function of immature CD8α＋ dentritic cells （DCs） in vitro. Methods The bone marrow and spleen of C57BL/6（H-2b） and Balb/c （H-2d） mice were got to prepare immature CD8α＋ DCs and spleen lymphocytes,and treated by mytomycin. MTT test was used.MLR group, MLR plus variable density syngeneic CD8α＋ DC group, MLR plus variable density allogeneic CD8α＋ DC group,MLR plus variable density CD8α＋ DC supernatant group,CD8α＋ DC plus syngeneic T cell group and negative control group were established. MLR group was set up by responder cell ratio of 0.2,0.5,0.8,1.0,to build the MLR plus syngeneic and allogeneic CD8α＋ DC experimental groups. Culture supernatant from different density （1 × 105/ml - 5 × 106/ml） of CD8α＋DCs was added into MLR to build CD8α＋ DC supernatant group. CD8α＋ DCs were co-cultured with syngeneic T cells to build CD8α＋ DCs plus syngeneic T cells group. 2 × 105/well responder cells served as the negative control group. ELISA was used to detect the concentrations of IFN-γ and IL-10 in the DCs could both suppress MLR （P〈0. 05）, and the difference was not statistically significant （P〉0. 05）. When CD8α＋ DCs were increased, the suppressive effect was enhanced. When CD8α＋ DC/responder cell ratio 〉0. 2, the inhibitory effect could be observed, and this effect reached the peak when the ratio was 1.0. The CD8α＋ DCs had weak ability to stimulate syngeneic lymphocyte proliferation in vitro, and certain stimulating effect could be seen only when CD8α＋ DC/responder cell ratio 〉2 （P〈0. 05）. Its culture supernatant also showed suppressive effect （P〈0. 05）, and the supernatant with a cell density of 5 × 105/ml showed the maximum effect. IL-10 concentration in the concentration was 1.0 ± 1.2 pg/ml. Conclusion The in vitro function of immature CD8α＋ DCs was immunosuppression/tolerance,and they could secret high level of IL-10. The CD8α＋ DCs and their culture supernatant could suppress MLR in vitro.

关 键 词：树突细胞 小鼠 淋巴细胞培养试验 混合 

分 类 号：R392[医药卫生—免疫学]