成熟与未成熟小鼠树突状细胞的生物免疫学特性研究

目的 研究小鼠骨髓成熟树突状细胞(mDC)与未成熟树突状细胞(imDC)的生物免疫学特性.方法 体外诱导培养小鼠骨髓imDC,经肿瘤坏死因子-α(TNF-α)刺激获得mDC.用小鼠CD11c免疫磁珠纯化DC.电镜观察DC的细胞形态.流式细胞术(FCM)检测细胞表面分子MHCⅡ、CD80、CD86的表达.用细胞增殖检测试剂(CCK-8)检测混合淋巴细胞反应(MLR)中DC刺激同种异基因T细胞的增殖能力.实时荧光定量PCR(QPCR)检测DC上细胞因子mRNA的表达.ELISA检测DC培养上清中的细胞因子表达.结果 电镜观察:imDC比mDC细胞突起少、短.胞质内有更多吞饮泡及溶酶体.FCM检测imDC上MHC-Ⅱ(27.2％)、CD80(27.6％)、CD86(29.5％)的表达水平均显著低于mDC MHC-Ⅱ(97.7％)、CD80(97.2％)、CD86(96.4％).MLR中相同反应比例imDC刺激T细胞的增殖能力,1∶5(1.63±0.04),1∶10(1.50±0.08),1∶20(1.28±0.07),1∶40(1.19±0.04),显著低于mDC 1∶5(2.21±0.09),1∶10(1.92 ±0.02),1∶20(1.64±0.01),1∶40(1.45±0.06),两组间差异均有统计学意义(均P＜0.01).QPCR检测imDC上IL12p35(0.66±0.13)、IL12p40(0.57±0.10)、IFN-γ(0.74±0.08)mRNA相对表达量(mDC为对照)显著低于mDC(1.00±0.00,P＜0.05),而TGF-β(1.35±0.09)显著高于mDC(1.00±0.00,P＜0.05).ELISA法检测imDC分泌IL12p70(6 ±4)、IFN-γ(56±15)显著低于mDC IL12p70(120±22)、I FN-γ(90±15,P＜0.05),而TGF-β(176±23)显著高于mDC TGF-β(55±18,P＜0.05).结论 imDC低表达共刺激分子、MHC-Ⅱ和Th1类细胞因子,高表达TGF-β,不能活化T细胞,具有耐受原性.mDC高表达共刺激分子、MHC-Ⅱ和Th1类细胞因子,可激活初始T细胞,具有免疫原性.

Bioimmunological characteristics of mature or immature murine dendritic cells

Objective To explore the biological characteristics of immature and mature murine bone marrow-derived dendritic cells(DCs).Methods The murine bone marrow cells were cultured and induced in vitro into immature DCs(imDCs).Then ImDCs were incubated with TNF(tumor necrosis factor)-αt to obtain mature DCs(mDCs).The DCs were purified by mutine CD11c microbeads.The morphologies of DCs were observed by electron microscopy(EM).Such surface markers as MHC-Ⅱ,CD80 and CD86 were tested by flow cytometry(FCM).The proliferation capacity of allogeneic T cells stimulated by DCs was examined by Cell Counting Kit-8(CCK-8)in mixed lymphocyte reaction(MLR).The expressions of cell factors in mRNA of DCs were tested by real-time polymerase chain reaction(QPCR).The cytokines in supernatant were measured by ELISA(enzyme-linked immunosorbent assay).Results As compared with mDCs,fewer,shorter spines and more phagocytic vesicles and lysosome were observed in imDCs under EM.The expressions of cell surface molecules in imDCs were significantly lower than those of mDC by FCM,MHC-Ⅱ(27.2％)vs(97.7％); CD80(27.6％)vs(97.2％); CD86(29.5％)vs(96.4％)].In MLR,the capacity of same-reaction ratio imDCs group for stimulating the proliferation of T-cells was remarkably lower than that of mDCs group,1∶ 5(1.63 ± 0.04)vs(2.21 ± 0.09); 1∶ 10(1.50 ± 0.08)vs(1.92 ±0.02); 1∶20(1.28 ±0.07)vs(1.64±0.01); 1∶40(1.19±0.04)vs(1.45±0.06),P＜0.01].In imDCs,the relative mRNA expressions of IL12p35(0.66 ±0.13),IL12p40(0.57 ±0.10)and IFN-γ (0.74 ± 0.08)were lower than those of mDCs(1.00 ± 0.00),(P ＜ 0.05),but the expression of TGF-β (1.35 ±0.09)was higher than that of mDCs by QPCR(1.00 ±0.00),(P＜0.05).The expressions of IL12p70 and IFN-γin supematants from imDCs were lower than those of mDCsIL12p70:(6 ±4)vs(120 ±22); IFN-γ:(56 ± 15)vs(90 ± 15),P ＜ 0.05]while the expression of TGF(transforming growth factor)-β was higher than that of mDCs by ELISATGF-β:(176 ± 23)vs(55 ± 18),P ＜0.05].Conclusion ImDCs can induce anergic T cells and immune tolerance through a down-regulation of costimulatory molecules,MHC-Ⅱ and Thl cytokines and an up-regulation of Th2 cytokine(TGF-β).And MDCs may activate naive T cells and stimulate immune responses by an up-regulation of co-stimulatory molecules,MHC-Ⅱ and Th1 cytokines.