致病性大肠杆菌感染小鼠肠炎模型的建立及其分子机制研究  

Establishment of Enteritis Model in Mice Infected with Enterotoxigenic Escherichia coli and Its Molecular Mechanism

作　　者：吴悦 王怡梦 王萌萌 周江涛 刘雪姣 李海花 乔家运 WU Yue;WANG Yimeng;WANG Mengmeng;ZHOU Jiangtao;LIU Xuejiao;LI Haihua;QIAO Jiayun(Tianjin Key Laboratory of Agriculture Animal Breeding and Healthy Husbandry,College of Animal Science and Animal Medicine,Tianjin Agricultural University,Tianjin 300384,China;Tianjin Key Laboratory of Conservation and Utilization of Animal Diversity,College of Life Sciences,Tianjin Normal University,Tianjin 300387,China)

机构地区：[1]天津农学院动物科学与动物医学学院,天津市农业动物繁育与健康养殖重点实验室,天津300384 [2]天津师范大学生命科学学院,天津市动物多样性保护与利用重点实验室,天津300387

出　　处：《动物营养学报》2021年第10期5817-5826,共10页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：天津市大学生创新训练计划项目(202010061007);天津市研究生科研创新项目(2020YJSS136);天津市“131”创新型人才团队(20180338)。

摘　　要：本试验旨在建立产肠毒素型大肠杆菌(ETEC)感染小鼠肠炎模型,并初步揭示其分子机制。将120只小鼠随机分为4组,每组30只,每组5个重复,每个重复6只。低、中、高剂量试验组分别经口腔灌服1×10^(5)、1×10^(6)和1×10^(7) CFU/只ETEC,对照组灌服等体积生理盐水,各组注射剂量均为0.2 mL。分别于攻毒后2、48、72、96和120 h观察小鼠临床表现并称量其体重,同时从每个组中随机取6只采集其血液和组织样本。采用酶联免疫吸附测定(ELISA)检测血清中C反应蛋白(CRP)、肿瘤坏死因子-α(TNF-α)和白细胞介素-8(IL-8)的含量,通过组织切片和苏木精-伊红(HE)染色法观察小鼠空肠、肝脏和脾脏的病理变化,利用实时荧光定量PCR检测Toll-样受体4(TLR4)、核因子kappa B p65亚基(NF-κB p 65)、髓样分化因子88(MyD88)、B细胞淋巴瘤因子3(BCl3)mRNA相对表达量。结果显示:与对照组相比,攻毒小鼠精神沉郁,进食减少,攻毒后72和96 h试验组小鼠体重均显著或极显著降低(P<0.01或P<0.05),肠道、肝脏和脾脏组织切片出现明显的炎症反应;攻毒后48和72 h试验组血清中CRP、TNF-α和IL-8的含量极显著升高(P<0.01);攻毒后48、72和96 h试验组空肠组织中TLR4、NF-κB p 65、MyD88 mRNA相对表达量极显著升高(P<0.01);攻毒后24和120 h试验组空肠组织中BCl3 mRNA相对表达量分别极显著或显著升高(P<0.01或P<0.05),攻毒后72 h呈极显著下降(P<0.01)。综上所述,本研究成功建立了ETEC感染小鼠肠炎模型,并且该病原菌可能是通过TLR4/NF-κB信号通路诱导炎症相关因子生成和组织中炎性细胞浸润,从而引起小鼠炎症反应。The aim of this study was to establish an enterotoxigenic Escherichia coli(ETEC)infection model of mouse enteritis,and to reveal its molecular mechanism.A total of 120 mice were randomly divided into 4 groups with 30 mice in each group,and there were 5 replicates per group and 6 replicates per replicate.The mice in low,medium and high dose experimental groups were given 1×10^(5),1×10^(6) and 1×10^(7) CFU/mice by gastric gavage,respectively,while those in control group was given equal volume of normal saline by gavage,with the injection dose of 0.2 mL in each group.Clinical manifestations of mice were observed and weighed at 24,48,72,96 and 120 h after challenge,respectively.Meanwhile,6 mice were randomly selected from each treatment group and their blood and tissue samples were collected.The contents of C-reactive protein(CRP),tumor necrosis factor-α(TNF-α),and interleukin-8(IL-8)in serum were detected by enzyme-linked immunosorbent assay(ELISA).The pathological changes of jejunum,liver and spleen of mice were observed by tissue sections and hematoxylin-eosin staining(HE staining).The mRNA relative expression levels of Toll-like receptor 4(TLR4),nuclear factor kappa-B(NF-κB p 65),myeloid differentiation factor 88(MyD88)and recombinant B-cell(BCl 3)were detected by fluorescence quantitative PCR.The results showed as follows:compared with the control group,challenged mice were depressed and eating less,the body weight of the three experimental groups was significantly decreased at 72 and 96 h after challenge(P<0.01 or P<0.05),intestinal,liver and spleen tissue sections showed obvious inflammatory reaction;the contents of CRP,TNF-αand IL-8 in serum of test groups were significantly increased at 48 and 72 h after challenge(P<0.01).The mRNA relative expression levels of TLR4,NF-κB p 65 and MyD88 in jejunum of experimental groups were significantly increased at 48,72 and 96 h after challenge(P<0.01).The mRNA relative expression levels of BCl3 in jejunum of test groups was significantly increased at 24 and 120 h

关 键 词：产肠毒素型大肠杆菌 TLR4/NF-κB信号通路 炎症反应 小鼠肠炎 

分 类 号：S853[农业科学—临床兽医学]