CAJ | 학술논문

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.
목적 관찰불성숙CD8α+수돌상세포(DC)적체외면역억제공능.방법 취C57BL/6(H-2b)소서화Balb/c(H-2d)소서적골수화비장,제비불성숙CD8α+DC화비림파세포,병경사렬매소처리.채용갑기새서기사서(MTT)법,실험설혼합림파세포반응(MLR)조(양성대조조)、MLR가불동밀도적동계CD8α+DC조、MLR가불동밀도동충이형CD8α+DC조、MLR가불동밀도CD8α+DC적배양상청조、CD8α+DC가동계T림파세포조급음성대조조.MLR조안자격세포/반응세포10∶1건립.CD8α+DC안여반응세포비례0.2∶1、0.5∶1、0.8∶1화1∶1적제도가입MLR중분별건립동계CD8α+D조화동충이형CD8α+D조;MLR중가입불동밀도(1×105/ml～5×106/ml)CD8α+DC적배양상청액건립CD8α+DC상청조;CD8α+DC여동계림파세포공배양건립CD8α+DC가동계T림파세포조;이반응세포2×105/공작위음성대조.채용매련면역흡부시험법검측MLR가동계CD8α+DC조(1∶1)상청액중γ간우소(IF-γ)화백세포개소10(IL-10)적농도.결과 동계화동충이형불성숙CD8α+DC대MLR균유억제작용(P＜0.05),이자간차이무통계학의의(P＞0.05).억제작용수DC비례적증가이증강,당CD8α+DC/반응세포비례대우0.2시현시억제작용(P＜0.05),비례위1시억제작용최강.CD8α+DC체외자격동계림파세포증식적능력교약,당DC여T림파세포적비치대우2시,현시출일정적자격작용(P＜0.05),기배양상청액대MLR야유억제작용(P＜0.05),기중밀도5×105/ml적세포배양상청액억제작용최강.MLR가동계CD8α+DC조(1∶1)상청액중IL-10적함량위(451.9±12.2)pg/ml,IFN-γ적함량위(1.0±1.2)pg/ml.결론 불성숙CD8α+DC체외구유면역억제혹유도면역내수적공능,가산생교고수평적IL-10,CD8α+DC급기배양상청액균가억제MLR.
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.