论文部分内容阅读
目的胰岛素抵抗(IR)及高胰岛素血症是动脉粥样硬化(AS)发生的重要危险因素,但其机制尚不明确。树突状细胞(Dendritic cell,DC)是目前发现的功能最强的专职性抗原提呈细胞(APC)。近年来研究发现,DC参与AS的免疫炎性反应过程。本研究探讨胰岛素对人单核源的树突状细胞(monocyte derired dendritic cells,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,100 ng／mL)和重组人白细胞介素-4(rhIL-4,20 ng／mL)的完全培养基中培养5 d,使其分化为MoDC,单独加入1、10和100 nmol／L的胰岛素。另用LY294002(P13K抑制剂)及PD98059(MAPK抑制剂)干预后再加1、10和100 nmol／L浓度的胰岛素,以PBS和LPS分别为阴性及阳性对照组,干预24 h后采用流式细胞术检测DC表型(CD83、CD86);FITC-Dextran检测DC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,10 nmol／L和100 nmol／L浓度的胰岛素明显上调MoDC表面CD83及共刺激分子CD86的表达(P<0．05),且10 nmol／L、100 nmol／L的胰岛素促进MoDC分泌细胞因子TNF-α、IFN-γ和IL-12,10 nmol/L的胰岛素减弱MoDC的吞噬功能,而1 nmol/L的胰岛素则无此作用。PD98059及LY294002干预后,10 nmol／L、100 nmol／L浓度胰岛素上调CD83及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)。结论高浓度胰岛素促进DC分化及免疫功能的成熟,这种作用是通过MAPK及P13K两条途经;高浓度胰岛素可能通过促进DC免疫功能成熟参与AS免疫炎性反应的发生、发展。
The purpose of insulin resistance (IR) and hyperinsulinemia is an important risk factor for atherosclerosis (AS), but the mechanism is not yet clear. Dendritic cells (DCs) are currently the most potent and specialized antigen-presenting cells (APC). In recent years, studies have found that DCs participate in the immune inflammatory reaction process of AS. This study was designed to investigate the effect of insulin on the differentiation and maturation of monocyte-derived dendritic cells (MoDC) and its immune mechanism. Methods Human peripheral blood mononuclear cells (CD14 +) were isolated by immunomagnetic beads method. The expression of CD14 + monocytes in human peripheral blood mononuclear cells (PBMCs) was detected with rhGM-CSF (100 ng / mL) and recombinant human interleukin- 20 ng / mL) for 5 days, differentiated into MoDC, and 1, 10 and 100 nmol / L of insulin were added separately. Another LY294002 (P13K inhibitor) and PD98059 (MAPK inhibitor) intervention after the addition of 1,10 and 100 nmol / L concentration of insulin, PBS and LPS were negative and positive control group, intervention 24 h after the use of streaming The DC phenotype (CD83, CD86) was detected by cytometry; the function of DC phagocytosis was detected by FITC-Dextran; the concentrations of cytokines IFN-γ, TNF-α and IL-12 were detected by ELISA. Results Compared with PBS, insulin at concentrations of 10 nmol / L and 100 nmol / L significantly upregulated the expression of CD83 and costimulatory molecule CD86 on MoDC (P <0.05), and promoted insulin at 10 nmol / L and 100 nmol / L MoDC secreted cytokines TNF-α, IFN-γ and IL-12, and 10 nmol / L insulin decreased phagocytosis of MoDC, while 1 nmol / L insulin did not. After the intervention of PD98059 and LY294002, the effects of 10 nmol / L and 100 nmol / L insulin up-regulating the expression of CD83 and CD86 and promoting the secretion of TNF-α, IFN-γ and IL-12 were significantly decreased (P <0.05). Conclusions High concentration of insulin can promote the differentiation of DC and the maturation of immune function through MAPK and P13K. High concentrations of insulin may participate in the development of immune and inflammatory reaction of AS through promoting the maturation of DC immune function.