摘要
本试验旨在通过丁酸梭菌(CB)、产肠毒素大肠杆菌(ETEC)和猪肠道上皮细胞(IPEC-J2细胞)共培养细胞模型,探讨CB对ETEC产黏附基因、产肠毒素基因以及ETEC刺激IPEC-J2细胞炎症相关因子表达的影响。试验设5组,分别为对照组、ETEC组、ETEC+CB 106组、ETEC+CB 107组、ETEC+CB 108组。ETEC组在IPEC-J2细胞培养液中加入1×108CFU/mL的ETEC,ETEC+CB 106组、ETEC+CB 107组、ETEC+CB 108组在IPEC-J2细胞培养液中加入1×108CFU/mL ETEC的同时分别加入1×106、1×107和1×108CFU/mL CB,对照组IPEC-J2细胞培养液中不添加ETEC和CB,37℃培养2 h后用显微镜观察细胞形态并收集细胞。采用实时定量PCR分析ETEC产黏附基因FaeG与肠毒素基因estA、estB及IPEC-J2细胞中促炎性因子白细胞介素(IL)-1β、IL-2、IL-6、IL-8与抗炎性因子IL-10 mRNA的相对表达水平。结果显示:CB可有效抑制ETEC诱导的IPEC-J2细胞损伤脱落。与ETEC组对比,不同浓度CB干预后显著抑制了ETEC产黏附基因FaeG与肠毒素基因estA、estB的表达(P<0.05),且CB浓度为1×108CFU/mL时效果最明显。与对照组相比,ETEC组IPEC-J2细胞中促炎性因子IL-1β、IL-2、IL-6和IL-8 mRNA的相对表达水平显著升高(P<0.05),抗炎性因子IL-10 mRNA的相对表达水平显著降低(P<0.05)。而添加不同浓度CB干预后,IPEC-J2细胞中IL-1β、IL-2、IL-6和IL-8 mRNA的相对表达水平较ETEC组显著降低(P<0.05),IL-10 mRNA的相对表达水平则较ETEC组显著升高(P<0.05),其中CB浓度为1×108CFU/mL时对IL-1β、IL-6和IL-8表达的抑制效果最强,浓度为1×107CFU/mL时对IL-2表达的抑制效果最强。以上结果表明,CB可降低ETEC黏附基因和产肠毒素基因表达,抑制ETEC诱导的猪肠道上皮细胞的炎症反应,降低ETEC对猪肠道上皮细胞的损伤作用。
The aim of this study was to investigate the effects of Clostridium butyrium(CB)on the expression of the adhesion and enterotoxin genes in entertoxigenic Escherichia coli(ETEC)and the inflammation-associated factors in porcine intestinal epithelial cells(IPEC-J2 cells)challenged by ETEC in a co-culture model of CB,ETEC and IPEC-J2 cells.The experiment included 5 groups,they were control group,ETEC group,ETEC+CB 106 group,ETEC+CB 107 group,ETEC+CB 108 group,respectively.In the ETEC group,IPEC-J2 cells were treated with 1×108 CFU/mL ETEC,while IPEC-J2 cells in ETEC+CB 106,ETEC+CB 107 and ETEC+CB 108 groups were treated with 1×108 CFU/mL ETEC 1×106,1×107,and 1×108 CFU/mL CB,respectively.Neither ETEC nor CB was added in the IPEC-J2 cells culture solution of the control group.Cell morphology was analyzed with microscope after incubation at 37℃for 2 h,and then cells were collected.The mRNA relative expression levels of adhesion gene FaeG and enterotoxigenic genes estA,estB in ETEC and pro-inflammatory factors interleukin(IL)-1β,IL-2,IL-6,IL-8 and anti-inflammatory factor IL-10 in IPEC-J2 cells were measured by quantitative real-time PCR(RT-qPCR).The results showed that CB could effectively inhibit the damage of IPEC-J2 cells induced by ETEC.Compared with ETEC group,different concentrations of CB treatment significantly reduced the expression of adhesion gene FaeG and enterotoxigenic genes estA,estB in ETEC(P<0.05),and the inhibitory effect was most obvious when the concentration of CB was 1×108 CFU/mL.Compared with the control group,the mRNA relative expression levels of pro-inflammatory factors IL-1β,IL-2,IL-6 and IL-8 were significantly increased(P<0.05),whereas the mRNA relative expression level of anti-inflammatory factor IL-10 was significantly decreased in IPEC-J2 cells treated by ETEC(P<0.05).However,the mRNA relative expression levels of IL-1β,IL-2,IL-6,and IL-8 were significantly decreased(P<0.05),but the mRNA relative expression level of IL-10 was significantly increased in IPEC-J2 cells by different concentrations of CB treatment compared with ETEC group(P<0.05).The inhibitory effects of CB on the expression of IL-1β,IL-6 and IL-8 were most effective when the concentration was 1×108 CFU/mL,and the inhibitory effects of CB on the expression of IL-2 was most effective when the concentration was 1×107 CFU/mL.These results suggest that CB can prevent the damage of porcine intestinal epithelial cells induced by ETEC through the inhibitory effects of the expression of adhesion and enterotoxigenic genes in ETEC and inflammatory reaction in porcine intestinal epithelial cells.
作者
杨华
施杏芬
桂国弘
吕文涛
李娜
肖英平
YANG Hua;SHI Xingfen;GUI Guohong;LYU Wentao;LI NaXIAO;Yingping(Institute of Quality and Standard for Agricultural Products,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;Zhejiang Institute of Veterinary Drug and Feed Control,Hangzhou 310018,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2019年第12期5688-5695,共8页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金项目(31402083)
金华市科学技术局农业类重点项目(2016-2-001)
关键词
丁酸梭菌
产肠毒素大肠杆菌
猪肠道上皮细胞
炎症反应
Clostridium butyrium
entertoxigenic Escherichia coli
porcine intestinal epithelial cells
inflammatory reaction
