脂多糖通过Galectin-9/Tim-3信号通路调控小鼠巨噬细胞M1/M2表型极化  

Effect of Galectin-9/Tim-3 pathway on the polarization of M1/M2 subtype in murine macrophages induced by lipopolysaccharide

作　　者：张望 章云涛[1] 方强[1] ZhangWang;ZhangYuntao;Fang Qiang(Department of Critical Care Medicine,the First Affiliated Hospital of Zhejiang University,Hangzhou 310003,Zhejiang,China)

机构地区：[1]浙江大学医学院附属第一医院重症医学科,浙江杭州310003

出　　处：《中华危重病急救医学》2018年第9期836-841,共6页Chinese Critical Care Medicine

基　　金：浙江省自然科学基金（LY16H150003）

摘　　要：目的探讨半乳糖凝集素- /T细胞免疫球蛋白黏蛋白-3（Galectin-/Tim-3）信号通路在脂多糖（LPS）诱导小鼠巨噬细胞M/2表型极化中的意义及分子机制。方法体外培养小鼠腹腔巨噬细胞RAW264.7，待细胞生长至80%~90%时用于实验。①将细胞以无血清DMEM培养基培养，并分别以0（空白对照）、0.01、0.1、1、10、100 m/的LPS刺激24 h。采用荧光定量反转录-聚合酶链反应（RT-qPCR）或蛋白质免疫印迹试验（Western Blot）检测巨噬细胞M1型标志物白细胞介素- 6（IL-6）、诱导型一氧化氮合酶（iNOS）和M2型标志物精氨酸酶-1（Arg-1）、白细胞分化抗原206（CD206）以及细胞内Tim-3、Galectin-9的表达。②另取小鼠腹腔巨噬细胞，并分为空白对照组（用无血清DMEM培养基培养24 h）、LPS处理组（用含0.1 m/的LPS无血清DMEM刺激24 h）和α-乳糖预处理组（在LPS刺激前1 h用含40 μmo/ Galectin-9信号拮抗剂α-乳糖的无血清DMEM进行预处理），通过封闭Galectin-9信号以验证Galectin-9在巨噬细胞M/2表型极化中的作用。结果①低浓度LPS（0.01 m/、0.1 m/）刺激24 h后，巨噬细胞M1型标志物的表达仅轻度上调（iNOS mRNA）或无明显变化（IL-6 mRNA）；而M2型标志物Arg-1 mRNA和CD206 mRNA表达则明显升高，并分别于0.1 m/和0.01 m/的LPS浓度下达到峰值〔与空白对照组比较：Arg-1 mRNA（2-ΔΔCt）为1.85±0.07比1.00±0.02，CD206 mRNA（2-ΔΔCt）为2.03±0.11比1.00±0.05，均P〈0.01〕；随LPS浓度升高，IL-6 mRNA和iNOS mRNA表达持续升高，而Arg-1 mRNA和CD206 mRNA表达则逐渐降低，巨噬细胞M/2表型极化状态发生改变。同时，经LPS刺激后，巨噬细胞内Tim-3蛋白表达在0.01 m/的LPS刺激后即较空白对照组明显上调（Tim-/APDH：0.84±0.04比0.69±0.02，P〈0.01），并在0.1 m/浓度下达峰值，之后随LPS浓度升高而逐渐下降；细胞内Galectin-9及上清中分泌型Galectin-9（s-Galectin-9）蛋白表�ObjectiveTo investigate the meaning and molecular mechanisms of Galectin-9/T-cell immunoglobulin mucin-3 （Tim-3） pathway on lipopolysaccharide （LPS） induced murine macrophage M1/M2 subtype polarization.MethodsThe murine peritoneal macrophages RAW264.7 were cultured in vitro until the cells had matured with 80%-90% fusion rate. ① The cells were cultured in serum-free medium and treated with 0 （blank control）, 0.01, 0.1, 1, 10 and 100 m/ LPS for 24 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction （RT-qPCR） or Western Blot was used to determine the expressions of M1 macrophage markers such as interleukin-6 （IL-6）, inducible nitric oxide synthase （iNOS） and M2 macrophage markers such as arginase-1 （Arg-1）, leukocyte differentiation antigen 206 （CD206）, as well as Tim-3 and Galectin-9 in the cells. ② The other mice peritoneal macrophages were divided into blank control group （cultured in serum-free DMEM medium for 24 hours）, LPS treatment group （cultured in serum-free DMEM medium containing 0.1 m/ LPS for 24 hours） and α-lactose pretreatment group （pretreated with serum-free DMEM containing 40 μmo/ Galectin-9 signal antagonist 1 hour before LPS stimulation）. Over closed Galectin-9 signal was used to verify the role of Galectin-9 in macrophage M/2 subtype polarization.Results① After stimulation with low concentrations of LPS （0.01 m/, 0.1 m/） for 24 hours, the expression of M1 markers was only slightly increased such as iNOS mRNA or not significantly changed such as IL-6 mRNA in macrophages, while the expressions of M2 markers such as Arg-1 mRNA and CD206 mRNA were significantly increased and peaked at LPS concentrations of 0.1 m/ and 0.01 m/ [compared with blank control group： Arg-1 mRNA （2-ΔΔCt） was 1.85±0.07 vs. 1.00±0.02, CD206 mRNA （2-ΔΔCt） was 2.03±0.11 vs.1.00±0.05, both P 〈 0.01]. With the increase of LPS concentration, the expressions of IL-6 mRNA and iNOS mRNA continued to increase, while the

关 键 词：半乳糖凝集素-9 T细胞免疫球蛋白黏蛋白-3 脂多糖 巨噬细胞表型 极化 

分 类 号：R459.7[医药卫生—急诊医学]