高糖对树突状细胞分化成熟和免疫功能的影响及其机制

Role of high glucose in maturation and immunologic function of dendritic cells

目的探索树突状细胞(DCs)在高糖环境下分化、成熟、免疫功能的影响及其发生机制,以期进一步探索DCs在糖尿病肾病发病中的作用.方法取小鼠骨髓单个核细胞,在含重组人粒?巨噬细胞集落刺激因子(rhGM-CSF,20 ng/ml)和重组人白细胞介素?4(rhIL-4,10 ng/ml)的完全培养基中培养,第7天收集不成熟DCs分别在含有5.5 mmol/L D-葡萄糖、30 mmol/L D?葡萄糖、30 mmol/L D-葡萄糖+100μg/ml核因子κB抑制剂吡咯烷二硫氨基甲酸(PDTC)的RPMI1640完全培养基中继续培养48 h后,采用流式细胞术检测DCs表型;以DCs为刺激细胞、同种异体T淋巴细胞为反应细胞按比例混合后,以混合淋巴细胞反应强度观察对树突状细胞抗原递呈、淋巴细胞增殖、淋巴细胞凋亡的影响;ELISA法检测细胞培养上清细胞因子浓度;Western blot检测核因子κB(NF-κB)活化程度.采用Student-t检验分析各组之间差异的显著性.结果与正常对照组相比,30 mmol/L D-葡萄糖能明显增加DCs内NF-κB的活化(IκBɑ:0.29±0.18比0.69±0.79,t=-1.27;p65:0.87±0.18比0.35±0.13,t=2.19;均P<0.01),显著上调DCs表面标志CD11c、主要组织相容性复合体Ⅱ类分子、CD80、CD86、CD40的表达(t=8.97~10.45,均P<0.05),增强了T淋巴细胞的增殖作用(1:100时1.80±0.23比1.57±0.20,t=2.46;1:25时1.74±0.17比1.32±0.18,t=2.82;1:10时1.59±0.25比1.28±0.19,t=3.06;均P<0.05)、减低了T淋巴细胞的凋亡比率(12.0±2比44.0±12.3,t=4.03,P<0.05),并且促进DCs分泌细胞因子白细胞介素12(IL-12)、γ-干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)(t=3.68~7.68,均P<0.05);而NF-κB抑制剂PDTC则可不同程度阻断上述作用(均P<0.05).结论高糖能够显著增加NF-κB的活化进而促进DCs的成熟,成熟DCs加速和放大了炎症免疫反应,这可能是DCs参与糖尿病肾病的重要机制之一.

Objective To explore the effect and mechanism of high glucose environmenton mouse bone marrow-derived dendritic cells′(DCs) differentiation, maturation, immune function, to further explore the role of DCs in inflammation of diabetic nephropathy. Methods Bone marrow mononuclear cells (MNCs) were isolated and cultured in the medium containing recombinant human granule macrophage colony stimulating factor (rhGM-CSF, 20 ng/ml) and recombinant human interleukin-4 (rhIL-4, 10 ng/ml). On 7th day, immature DCs were collected and cultured in RPMI1640 complete medium containing 5.5 mmol/L D-glucose, 30 mmol/L D-glucose, 30 mmol/L D-glucose+100 μg/ml NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) for 48 h, and then flow cytometry (FCM) was used to detect the phenotype of DCs. Cells were stimulated with DCs, allogeneic T lymphocytes were mixed in proportion and the reaction intensity of mixed lymphocytes was used to observe the effect on dendritic cell antigen presentation, lymphocyte proliferation, lymphocyte apoptosis. ELISA assay was adopted to detect cytokines concentration of cell culture supernatant. Western blot was adopted for detection of NF-κB activation. Student-t test was used to analyze the significance of the differences between the groups. Results Compared with the normal control group, 30 mmol/L D-glucose significantly increased the activation of NF-κB in DCs (IκBɑ:0.29±0.18 vs 0.69±0.79, t=-1.27;p65: 0.87±0.18 vs 0.35±0.13, t=2.19;all P<0.01) and the expression of eCD11c, MHC class Ⅱ molecules, CD80, CD86 and CD40 on DCs (t=8.97-10.45, all P<0.05). It promoted the T lymphocyte proliferation (1∶100:1.80±0.23 vs 1.57±0.20, t=2.46;1∶25:1.74±0.17 vs 1.32±0.18, t=2.82;1∶10:1.59±0.25 vs 1.28±0.19, t=3.06;all P<0.05), weakened the T lymphocyte apoptosis rate (12±2 vs 44±13, t=4.03, P<0.05). And it promoted DCs′ secretion of cytokines IL-12, IFN-γ and TNF-α(t=3.68-7.68, all P<0.05), while the inhibition of NF-κB agent PDTC could block the above effects to varying degrees (all P<0.05). Conclusion High glucose can significantly increase the activation of NF-κB thereby promote the maturation of DCs, which accelerates and amplifies the inflammatory response. This may be one of the important mechanisms of dendritic cells in diabetic nephropathy.