高糖对人单核细胞源树突状细胞分化成熟和免疫功能的影响及其机制研究  

Effects and mechanism of hyperglycemia on development and maturation and immune function of human monocyte derived dendritic cells

作　　者：姚康[1] 葛均波[1] 孙爱军[1] 洪晓武[2] 施鸿毓[1] 黄榕翀[1] 贾庆哲[1] 王克强[1] 钟翠平[3] 曹雪涛[4] 邹云增[1] 

机构地区：[1]上海市心血管病研究所复旦大学附属中山医院心内科,200032 [2]复旦大学上海医学院免疫学中心 [3]复旦大学上海医学院解剖与组织胚胎学系 [4]第二军医大学免疫学研究所全军免疫与基因治疗重点实验室

出　　处：《中华心血管病杂志》2006年第1期60-64,共5页Chinese Journal of Cardiology

基　　金：国家重点基础研究发展规划资助项目(G2000056903);上海市科委资助项目(02JC14026)

摘　　要：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 Dendritic cells play an important role in the pathogenesis of atherosclerosis. To explore the effects of hyperglycemia on the maturation and immune function of human monocyte derived dendritic cells （MDCs）. Methods Immature MDCs were cultured in RPMI1640 medium with either 5.5 mmol/L D-glucose （NG）, 25 mmoL/L D-glucose（HG） or 5.5 mmol/L D-glucose ＋ 19. 5 mmol/L mannitol （HM） in the absence or presence of 30 mmol/L N-acetyleysteine[NAC, a reactive oxygen species inhibitor （ROS）] for 48 hours. FACS was used to investigate the MDCs immunophenotypic expression. Immune function was evaluated by allogeneic mixed T lymphocyte reaction and measurement of cytokine levels from culture supematants. Intracellular ROS production in MDCs was also measured by 2′, 7′- dichlorodihydrofluorescin （DCF, 10μmoL/L） fluorescence using confocal laser-scanning microscopy techniques. Results Compared with NG and HM treated MDCs, the expression of maturation markers such as CDla, HLA-DR, CD83, CD86 were significantly upregulated, allogeneic T cells proliferation as well as the cytokines secretions （IL-2, IL-12, IL-10 and IFN-γ） significantly increased in HG treated MDCs. Intracellular ROS production in MDCs was also significantly increased and all these stimulatory effects of HG could be partially attenuated by NAC. Conclusion High glucose promote the maturation of MDCs and augment their capacity to stimulate T-cell proliferation and cytokine secretions at least in part through enhancing intracellular ROS generation. These stimulating effects of high glucose on MDCs maturation may be one of the mechanisms of accelerated atherosclerosis found in patients with diabetes.

关 键 词：动脉硬化 糖尿病 自身免疫 树突细胞 

分 类 号：R543.5[医药卫生—心血管疾病]