高浓度胰岛素对树突状细胞分化成熟及免疫功能的影响

目的胰岛素抵抗及高胰岛素血症是动脉粥样硬化发生的重要危险因素,但其机制尚不明确。树突状细胞是目前发现的功能最强的专职性抗原提呈细胞,研究发现树突状细胞参与了动脉粥样硬化免疫炎症反应的过程。研究探讨胰岛素对人单核源的树突状细胞（monocytederiveddendriticcell,MoDC）分化成熟、免疫功能的影响及其作用机制。方法采用免疫磁珠法分离人外周血CD14^＋单核细胞,在含重组人粒．单核细胞集落刺激因子（rhGM—CSF,100μg／L）和重组人白细胞介素-4（rhIL-4,20μg／L）的完全培养基中培养5天,使其分化为MoDC。然后单独加入1、10、100nmol／L浓度的胰岛素。另用P13K抑制剂LY294002及MAPK抑制剂PD98059干预后再加入1、10、100nmol／L浓度的胰岛素。以PBS和脂多糖（LPS）分别为阴性及阳性对照组。干预24h后采用流式细胞术检测树突状细胞表型（CD83、CD86）。FITC—Dextran检测树突状细胞吞噬功能。ELISA法检测细胞培养上清细胞因子（IFN—γ、TNF-α和IL．12）浓度。结果与PBS对照组比较,10、100nmol／L浓度的胰岛素明显上调了MoDC表面CD83及共刺激分子CD86的表达（P〈0．05）,且促进MoDC分泌细胞因子TNF-α、IFN-γ和IL-12,10nmol／L的胰岛素减弱了MoDC的吞噬功能,而1nmol／L的胰岛素则没有以上作用。PD98059及LY294002干预后使10、100nmol／L浓度胰岛素上调CD83和CD86表达及促进TNF-α、IFN-γ和IL-12分泌的作用明显减弱（P〈0．05）。结论高浓度胰岛素通过MAPK及P13K两条途径促进了树突状细胞分化及免疫功能的成熟。高浓度胰岛素可能通过促进树突状细胞免疫功能成熟参与了动脉粥样硬化免疫炎症反应的发生、发展。

Hyperinsulinemia induced immune maturation of human monocyte derived dendritic cells: bridging between diabetes and atherosclerosis

OBJECTIVE: Dendritic cells an hyperinsulinemia are both implicated in the pathogenesis of atherosclerosis. The aim of this study is to explore the effect of high concentration of insulin on the maturation of monocyte-derived dendritic cells (MoDCs) and related signal transduction pathways. METHODS: Human monocytes were purified (over 98%) using Anti-CD14 micro-beads and cultured for 5 days with DC Cellgro medium containing rhGM-CSF (100 microg/L) and rhIL-4 (20 microg/L). Immature DC were then incubated with insulin of various concentrations (0, 1, 10, 100 nmol/L) for 24 hours in the presence or absence of LY294002 (PI3K inhibitor) or PD98059 (MAPK inhibitor). Immunophenotypic expression of CD86 and CD83 were detected using flow cytometry. Endocytosis function of the MoDCs was evaluated using FITC-Dextran and MoDCs secretion IL-12, IFN-gamma and TNF-alpha were measured by ELISA. RESULTS: Insulin induced significantly higher CD83 and CD86 expressions on MoDCs in a dose-dependent manner. The endocytosis function of MoDCs were significantly inhibited and cytokine secretions of IL-12, IFN-gamma and TNF-alpha significantly increased by 10 nmol/L and 100 nmol/L insulin. These effects could be blocked by the LY294002 and PD98059. CONCLUSION: Hyperinsulinemia contributed to atherosclerosis via stimulating immune maturation of MoDCs via both PI3K and MAPK pathways.