Toll样受体4接头分子对乳酸诱导巨噬细胞M2极化的促进作用及其机制  

Effect of adapters of Toll-like receptor 4 on M2 polarization of macrophages induced by lactate and its mechanism

作　　者：陈为[1,2] 沈楠[2] 韩宛娜[2] 郗艳丽[3] 任旷[2] 金连海[2] 许娜[1] CHEN Wei;SHEN Nan;HAN Wanna;XI Yanli;REN Kuang;JIN Lianhai;XU Na(Platform of Biomedical Innovating and Transforming,Jilin Medical University,Jilin 132013,China;Center of Hypobaric Hypoxic Environmental Intervening and Innovating,Jilin Medical University,Jilin 132013,China;Department of Toxicology,Jilin Medical University,Jilin 132013,China)

机构地区：[1]吉林医药学院生物医药双创转化实训平台,吉林吉林132013 [2]吉林医药学院低压低氧环境与健康干预创新中心,吉林吉林132013 [3]吉林医药学院毒理学教研室,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2022年第5期1190-1199,共10页Journal of Jilin University：Medicine Edition

基　　金：吉林省教育厅科学研究项目(JJKH20200457KJ);吉林省卫健委青年科技骨干培养计划项目(2019Q033)。

摘　　要：目的:探讨Toll样受体4 (TLR4)接头分子髓样分化因子88 (MyD88)和诱导β干扰素的Toll/白细胞介素1 (IL)受体(TIR)结构域衔接蛋白(TRIF)基因敲除后对乳酸诱导巨噬细胞极化的促进作用,初步阐明乳酸免疫调控的可能机制。方法:体外培养小鼠巨噬细胞Raw264.7,采用慢病毒法构建CRISPR/Cas9基因编辑载体,定向敲除MyD88和TRIF基因(MyD88-KO组和TRIF-KO组),设未感染的Raw264.7细胞为对照组,实时荧光定量PCR (RT-qPCR)法和Western blotting法检测基因敲除效果。实验分为未处理Raw264.7细胞组、Raw264.7+乳酸组、MyD88-KO组、MyD88-KO+乳酸组、TRIF-KO组和TRIF-KO+乳酸组。15 mmol·L^(-1)乳酸诱导24 h后进行各指标检测。RT-qPCR法检测各组细胞中M2极化表型分子巨噬细胞甘露糖受体CD206和精氨酸酶1(Arg1) mRNA表达水平,光学显微镜下观察各组细胞形态表现,酶联免疫吸附测定(ELISA)法检测各组细胞上清中肿瘤坏死因子α (TNF-α)、γ干扰素(INF-γ)和白细胞介素10 (IL0)水平,Western blotting法检测各组细胞中核因子κB (NF-κB)信号通路相关蛋白表达水平。采用Autodock软件将乳酸分别与TLR4、MyD88和TRIF分子对接并观察其结合情况。结果:采用CRISPR/Cas9技术进行基因定向敲除后,与对照组比较,MyD88-KO组和TRIF-KO组细胞中MyD88和TRIF mRNA及蛋白表达水平明显降低(P<0.05或P<0.01)。乳酸作用24 h后,与未处理Raw264.7细胞组比较,Raw264.7+乳酸组细胞中M2极化标志物CD206和Arg1 mRNA表达水平明显升高(P<0.05);与MyD88-KO组比较,MyD88-KO+乳酸组细胞中CD206和Arg1 mRNA表达水平均升高(P<0.05);与TRIF-KO组比较,TRIF-KO+乳酸组细胞中CD206和Arg1 mRNA表达水平均降低(P<0.05)。细胞形态表现,Raw264.7+乳酸组细胞表现出M2极化形态,即体积增大、明显突起伪足和偏锥形细胞比例增加;MyD88-KO+乳酸组细胞同样表现出M2极化形态变化;TRIF-KO+乳酸组细胞形态变化不明显。ELISA法检测,与未处理Raw2Objective:To investigate the promotion effect of Toll-like receptor 4(TLR4) adapters,myeloid differentiation factor 88(MyD88)and Toll/interleukin(IL)receptor(TIR)domain containing adaptor protein inducing interferon-β(TRIF) after gene knowout in M2 polarization of macrophages induced by lactate, and to elucidate the regulation mechanism of immune responses by lactate.Methods:The murine macrophage cell line Raw264. 7 were cultured in vitro,CRISPR/Cas9 gene editing system was constructed by lentivirus method,MyD88 and TRIF genes were knocked out(Myd88-KO group and TRIF-KO group),and the untransfection Raw264. 7 cells were used as control group. The knockout effects of gene knowout were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods. The experiment was divided into untreated Raw264. 7 cell group,Raw264. 7 + lactate group,MyD88-KO group,MyD88-KO + lactate group,TRIF-KO group and TRIF-KO+lactate group. The indexes were determined after 15 mmol·L^(-1)lactate were induction for 24 h.The expression levels of M2 polarization phenotype molecules macrophage mannose receptor CD206 and arginase 1(Arg1) mRNA in the cells in various groups were detected by RT-qPCR method, the morphology of cells in various groups was observed under light microscope,and the levels of tumor necrosis factor-α(TNF-α),interferon-γ(INF-γ) and interleukin0(IL0) in the cell supernatant in various groups were detected by enzyme linked immunosorbent assay(ELISA)method. The expression levels of nuclear factor-κB(NF-κB)signaling pathway-related proteins were detected by Western blotting method.The molecular docking simulation of lactate with TLR4,MyD88 and TRIF were carried out by Autodock software,and the binding was observed. Results:After targeted gene was knocked out using CRISPR/Cas9technique,compared with control group,the expression levels of MyD88 and TRIF mRNA and proteins in the cells in MyD88-KO group and TRIF-KO group were significantly reduced(P<0. 05 or P<0. 01).After 24 h of lactate treatment

关 键 词：TOLL样受体4 髓样分化因子88 诱导β干扰素的TIR结构域衔接蛋白 乳酸 巨噬细胞 

分 类 号：R392.12[医药卫生—免疫学]