高浓度胰岛素对树突状细胞的凝集素样氧化表达的影响及其机制  

Effect and mechanism of high concentration of insulin on the expression of lectin-like oxidized lowdensity lipoprotein receptor 1 in dendritic cells

作　　者：陆浩[1] 姚康[1] 黄东[1] 李晨光[1] 常书福[1] 戴宇翔[1] 孙爱军[1] 邹云增[1] 钱菊英[1] 葛均波[1] 

机构地区：[1]复旦大学附属中山医院心内科,上海市心血管病研究所,上海200032

出　　处：《上海医学》2015年第5期421-425,共5页Shanghai Medical Journal

基　　金：国家自然科学基金资助项目(81400318)

摘　　要：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 explore the effect of high concentration of insulin on the expression of lectin- like oxidized low-density lipoprotein receptor-1 （LOX-1） in dendritic cells （DC）. Methods Peripheral blood mononuclear cells were purified by using CD14^＋ immunomagnetic micrebeads and incubated in serum free culture medium for cryepreservatian （RPMI-1640） medium supplemented with recombinant human granulocyte macrophage colony stimulating factor（GM-OSF） 100 ng/mL and IL-4 （50 ng/mL） for 5 days. Immature DC were cultured with different concentrations of insulin （1, 10, 50 and 100 nmol/L）. In some experiments, phosphatidylinositol-3-OH kinase （PI3K） pathways inhibiter （LY294002） or mitogen activated protein kinase （MAPK） inhibitor （PD98059） was added simultaneously with 100 nmol/L insulin for 24 h. The expression of LOX-1 was determined by real-time polymerase chain reaction （PCR） and Western blotting analysis. Furthermore, DO were pretreated with 1 nmol/L insulin, 100 nmol/L insulin or 100 nmol/L insulin -t- anti-LOX-1 neutralizing antibodies for 24 h, then DO were incubated with Dil-labelled oxLDL for another 4 h. The Dil-oxLDL incorporated fraction was investigated by flow cytometry. Results Compared with 1 nmol/L insulin culture, the incubation of DO with 10 nmol/L, 50 nmol/L and 100 nmol/L insulin significantly enhanced LOX-1 gene and protein expression （all P〈0.05）. The expression of LOX-1 was in a dose-dependent manner in the last three groups （both P〈0.05）. The expression of LOX-1 mRNA and protein in PD98059＋ 100 nmol/L insulin group was significantly lower than those in 100 nmol/L insulin group （both P〈0. 05）. There was no statistical difference in the expression of LOX-1 mRNA and protein between LY294002＋ 100 nmol/L insulin group and 100 nmol/L insulin group （both P〉0.05）. Compared with 1 nmol/L insulin culture, 100 nmol/L insulin increased the oxLDL-uptake capacity of DO by 138.2% ---23.1% （P〈0.05）. The uptake of oxLDL decreasedly 4

关 键 词：胰岛素 树突状细胞 动脉粥样硬化 2型糖尿病 低密度脂蛋白受体-1 

分 类 号：R587.2[医药卫生—内分泌] R543.5[医药卫生—内科学]