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目的探讨高浓度胰岛素对树突状细胞(DC)的凝集素样氧化低密度脂蛋白受体-1(LOX-1)表达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,经含重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)100ng/mL和重组人IL-4(rhIL-4)50ng/mL的无血清细胞冻存培养基(RPMI-1640培养基)培养液培养5d,使其分化为未成熟DC。分别加入终浓度为1(胰岛素1组)、10(胰岛素10组)、50(胰岛素50组)、100nmol/L(胰岛素100组)的胰岛素,另用磷脂酰肌醇3激酶(PI3K)抑制剂(LY294002,LY294002+胰岛素100组)和丝裂原激活蛋白激酶(MAPK)抑制剂(PD98059,PD98059+胰岛素100组)干预后再加入100nmol/L的胰岛素,干预24h后,收集细胞。采用实时荧光定量PCR(RT-PCR)和Western印迹法检测LOX-1蛋白表达。同时,用1、100nmol/L的胰岛素干预DC 24h后,加入LOX-1阻断抗体(抗LOX-1+胰岛素100组),将DC与荧光DiI标记的oxLDL(DiI-oxLDL)共同孵育4h,采用流式细胞术检测DC吞噬oxLDL情况。结果胰岛素10组、胰岛素50组、胰岛素100组的LOX-1mRNA、蛋白表达相对比值均显著高于胰岛素1组(P值均<0.05),前3组间的差异有统计学意义(P值均<0.05),且呈浓度依赖性。PD98059+胰岛素100组的LOX-1mRNA、蛋白表达相对比值均显著低于胰岛素100组(P值均<0.05),LY294002+胰岛素100组与胰岛素100组间的差异无统计学意义(P值均>0.05)。与胰岛素1组相比,胰岛素100组的DC摄取DiI-oxDL的能力增加了(138.2±23.1)%,两组间差异有统计学意义(P<0.05);而抗LOX-1+胰岛素100组摄取的DiI-oxLDL较胰岛素100组下降了(48.9±12.9)%,两组间差异有统计学意义(P<0.05)。结论高浓度胰岛素明显上调DC的LOX-1的表达,该作用主要是通过MAPK信号通路起作用,PI3K信号通路在其中并不起主要作用,而且高浓度胰岛素通过LOX-1促进DC摄取oxLDL。
Objective To investigate the effect of high concentration insulin on the expression of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) in dendritic cells (DCs) and its mechanism. Methods Human peripheral blood mononuclear cells (CD14 +) were isolated by immunomagnetic beads method. The cells were transfected with 100ng / mL recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and 50ng / mL recombinant human IL- Of serum-free cell freezing medium (RPMI-1640 medium) culture medium for 5 days, it was differentiated into immature DC. The cells were treated with insulin (group 1), insulin (group 10), insulin (group 50) and insulin (100nmol / L) respectively, and then treated with phosphatidylinositol 3 kinase (PI3K) (LY294002, LY294002 + insulin group 100) and mitogen-activated protein kinase (MAPK) inhibitor (PD98059, PD98059 + insulin group 100) were added to 100nmol / L insulin. After 24h intervention, cells were harvested. LOX-1 protein expression was detected by real-time fluorescence quantitative PCR (RT-PCR) and Western blotting. At the same time, DCs were exposed to 1,100 nmol / L of insulin for 24 h. LOX-1 blocking antibody (anti-LOX-1 + insulin 100) was added and DCs were incubated with fluorescent DiI labeled oxLDL (DiI-oxLDL) Flow cytometry was used to detect the condition of oxLDL engulfed by DC. Results The relative ratios of LOX-1mRNA and protein expression in 10 groups of insulin, 50 groups of insulin and 100 groups of insulin were significantly higher than those in 1 group (all P <0.05), the differences among the three groups were statistically significant <0.05), and in a concentration-dependent manner. The relative ratio of LOX-1 mRNA and protein expression in PD98059 + insulin group was significantly lower than that in insulin group 100 (all P <0.05). There was no significant difference between LY294002 + insulin group and insulin group (P> 0.05) . Compared with insulin group 1, the ability of DC to take in DiI-oxDL in insulin group increased by (138.2 ± 23.1)%, with significant difference between the two groups (P <0.05); while the resistance to LOX-1 + insulin group The DiI-oxLDL uptake was decreased by (48.9 ± 12.9)% compared with insulin 100 group, with a significant difference between the two groups (P <0.05). CONCLUSION: High concentration of insulin significantly up-regulates the expression of LOX-1 in DC. The effect is mainly through the MAPK signaling pathway, in which PI3K signaling pathway does not play a major role, and high concentrations of insulin promote DC uptake of oxLDL by LOX-1.