非洲猪瘟病毒感染过程中巨噬细胞极化表型变化的研究  

Study on the polarization phenotype changes of macrophage during African swine fever virus infection

作　　者：崔宏全 姜成刚[1] 文莉莉 范宇琴 刘英豪 都兰 王靖飞[1] 赵东明 何希君[1] CUI Hong-quan;JIANG Cheng-gang;WEN Li-i;FAN Yu-qin;LIU Ying-hao;DU Lan;WANG Jing-fei;ZHAO Dong-ming;HE Xi-jun(State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2025年第1期54-62,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2021YFD1801405)。

摘　　要：为研究非洲猪瘟病毒(ASFV)对巨噬细胞极化表型的影响,本研究将100ng/mL脂多糖(LPS)+50ng/mL IFN-γ诱导猪肺泡巨噬细胞(PAM)成为M1型巨噬细胞(M1组),用50 ng/mL IL-4诱导PAM成为M2型巨噬细胞(M2组),以不做任何处理的PAM作为对照组。24 h后采用荧光定量PCR(qPCR)检测各组细胞中M1型细胞因子IL-1β、IL-6、IL-12、TNF-α及M2型细胞因子IL-10及其标志物精氨酸酶-1(Arg-1)、几丁质酶-1(Ym-1)、过氧化物酶体增殖物激活受体(PPAR)mRNA的转录水平;采用Luminex细胞多因子试剂盒检测各组细胞上清中M1型细胞因子TNF-α、IL-1α、IL-1β、IL-6、IL-12、IL-18和M2型细胞因子IL-1受体拮抗剂(IL-1RA)、IL-10的表达水平;采用NO试剂盒及流式细胞术分别检测各组细胞上清中M1型巨噬细胞代谢物NO的含量及其细胞表面标志物CD80+细胞的占比;采用western blot检测各组细胞中M2型巨噬细胞标志物Arg-1蛋白的表达。结果显示,M1组中促炎细胞因子IL-1β、IL-6、IL-12、TNF-αmRNA的转录和蛋白分泌水平、IL-1α的分泌水平、NO的含量及CD80+细胞的占比均显著和极显著高于其余两组。M2组中抑炎细胞因子IL-10和Arg-1 mRNA的转录和蛋白的分泌水平均极显著高于其余两组。未检测到IL-18和IL-1RA的表达。在此基础上将ASFV Pig/HLJ/2018株以MOI 1感染PAM,不同时间后(0、12 h、24 h、36 h及48 h)分别按照上述各方法检测PAM中各细胞因子及极化标志物的转录水平及蛋白的分泌水平,分析ASFV感染后巨噬细胞表型的极化方向。结果显示,与对照组相比,ASFV感染后PAM中M1型细胞因子IL-1β、TNF-α、IL-6的转录水平均极显著升高,IL-6的转录水平一直维持在较高水平,PAM中M2型细胞因子IL-10及Arg-1的转录水平先升后降,并分别于感染后48h与12 h达到峰值,后者随后下降;Ym-1与PPAR的转录水平均在感染后24 h达到峰值,随后下降;ASFV感染后PAM中部分细胞因子蛋白的分泌水平与其转录水平的This study aimed to explore the influence of African swine fever virus(ASFV)on the polarization phenotype of macrophages.To achieve this,porcine alveolar macrophages(PAM)were induced to differentiate into M1 macrophages(M1 group)using 100ng/mL lipopolysaccharide(LPS)+50ng/mL interferon-γ(IFN-γ),into M2 macrophages(M2 group)using 50ng/mL interleukin-4(IL-4),and untreated PAM served as the control group.After 24 hours of induction,fluorescence quantitative PCR(qPCR)was employed to assess the transcription levels of M1 cytokines(IL-1β,IL-6,IL-12,TNF-α)and M2 cytokines(IL-10),along with markers such as arginase-1(Arg-1),chitinase 1(Ym-1),peroxisome proliferator-activated receptor(PPAR).The expression levels of M1 cytokines TNF-α,interleukin-1α(IL-1α),IL-1β,IL-6,IL-12,interleukin-18(IL-18),and M2 cytokines IL-1 receptor antagonist(IL-1RA),IL-10 in cell supernatants were detected using a Luminex cell multi-factor detection kit;Nitric oxide(NO)content in cell supernatant and proportion of polarization marker CD80+cells in M1 macrophages was measured using a kit and flow cytometry,respectively.Western blot was conducted to detect the expression of the M2 macrophage marker Arg-1 protein.Results indicated that the transcription and protein secretion levels of pro-inflammatory cytokines IL-1β,IL-6,IL-12,TNF-α,the secretion level of IL-1α,NO content,as well as the proportion of CD80+cells in the M1 group,were significantly higher than those in the other two groups.Conversely,the transcription and protein secretion levels of anti-inflammatory cytokines IL-10 and Arg-1,were notably higher in the M2 group.IL-18 and IL-1RA were undetectable.Subsequently,PAM were infected with ASFV(Pig/HLJ/2018 strain)at an MOI of 1,and the transcription and protein expression levels of various cytokines and polarization markers were detected at different time points(0,12 hours,24 hours,36 hours,and 48 hours)using the methods mentioned above.The result revealed that compared to the control group,the transcription levels of M1 cytokines

关 键 词：非洲猪瘟病毒 肺泡巨噬细胞 极化 

分 类 号：S852.65[农业科学—基础兽医学]