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目的探讨高糖对人单核细胞源的树突状细胞(monocyte deriveddendritic cells,MDCs)的分化、成熟及其免疫功能的影响,进一步探索高糖在动脉粥样硬化免疫炎症反应中的作用。方法免疫磁珠法分离人外周血CD14+单核细胞,在含重组人粒-巨噬细胞集落刺激因子(rhGM-CSF,100ng/ml)和重组人白细胞介素-4(rhIL-4,20ng/ml)的完全培养基中培养;不成熟MDCs在含有5·5mmol/LD-葡萄糖、25mmol/LD-葡萄糖、5·5mmol/LD-葡萄糖+19·5mmol/L甘露醇以及25mmol/LD-葡萄糖+30mmol/LN-乙酰半胱氨酸(N-acetyleysteine,NAC)RPMI1640完全培养基中继续培养48h后,采用流式细胞术检测MDCs表型;以MDCs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和淋巴细胞增殖的影响;ELISA法检测细胞培养上清细胞因子浓度;利用荧光探针还原型二氯荧光素(2′,7′-dichlorodihydrofluorescin,DCFH)在共聚焦显微镜下检测MDCs内活性氧水平(reactiveoxygenspecies,ROS)。结果与正常对照组比较,25mmol/LD-葡萄糖明显增强MDCs内ROS水平(P<0·01),显著上调MDCs表面标志CD86、CD83、CD1a、HLA-DR表达,增强T淋巴细胞增殖作用(P<0·01)并且促进MDCs分泌细胞因子IFN-γ、IL-10、IL-12、IL-2(P<0·05);而ROS抑制剂NAC可部分阻断高糖的上述作用。结论高糖显著增强树突状细胞内ROS,从而促进树突状细胞成熟,激活T淋巴细胞的能力也明显增强,并且通过促进树突状细胞本身释放一些炎症因子,加速和放大炎症免疫反应。这可能是树突状细胞参与动脉粥样硬化炎症免疫反应发生的重要机制之一。
Objective To investigate the effect of high glucose on the differentiation, maturation and immune function of monocyte derived dendritic cells (MDCs), and to further explore the role of high glucose in the immune inflammatory reaction of atherosclerosis. Methods Human peripheral blood mononuclear cells (CD14 +) were isolated by immunomagnetic beads method and cultured in the medium containing rhGM-CSF (100ng / ml) and recombinant human interleukin-4 (20ng / ml). Immature MDCs were cultured in medium containing 5.5 mmol / L-glucose, 25 mmol / L-glucose, 5.5 mmol / L-glucose and 19.5 mmol / L mannitol and 25 mmol / After cultured for 48h in RPMI1640 complete medium containing 30mmol / L NAC (acetylcysteine, NAC), the phenotypes of MDCs were detected by flow cytometry. The cells were stimulated with MDCs and allogeneic T lymphocytes reacted After the cells were mixed in proportion, the effect of mixed lymphocyte reaction intensity on the dendritic cell antigen presentation and lymphocyte proliferation was observed; the cytokine concentration in the supernatant of the cell culture was detected by ELISA; the fluorescence intensity of dichlorofluorescein 2 ’, 7’-dichlorodihydrofluorescin, DCFH) to detect reactive oxygen species (ROS) in MDCs under confocal microscope. Results Compared with the normal control group, 25mmol / L-glucose significantly increased the level of ROS in MDCs (P <0.01), significantly upregulated the expression of CD86, CD83, CD1a and HLA-DR on MDS surface and increased the proliferation of T lymphocytes <0.01) and promoted the secretion of cytokines IFN-γ, IL-10, IL-12 and IL-2 (P <0.05) by MDCs. The ROS inhibitor NAC partially blocked the above effects of high glucose. CONCLUSION: High glucose can significantly increase ROS in dendritic cells, promote the maturation of dendritic cells, enhance the ability of activating T lymphocytes, and accelerate and amplify the inflammatory response by promoting the release of inflammatory cytokines by dendritic cells. This may be one of the important mechanisms that dendritic cells take part in the atherosclerotic inflammatory reaction.