致病性大肠杆菌HPI对Caspase-1细胞焦亡相关分子表达的影响

doi:10.16431/j.cnki.1671-7236.2019.11.006

摘要/Abstract

摘要： 为研究云南撒坝猪致病性大肠杆菌高致病性毒力岛（HPI）致猪源巨噬细胞焦亡的分子机制，本试验以云南撒坝猪致病性大肠杆菌HPI阳性株感染猪源巨噬细胞为切入点，从云南楚雄州某规模养殖场采集撒坝猪仔猪黄白痢的粪便，对大肠杆菌进行分离纯化，并通过PCR技术对HPI irp2基因进行检测，分别以HPI阳性株（HPI⁺）和阴性株（HPI^-）感染巨噬细胞，并设立LPS+ATP组和空白对照组，于0.5和6 h收集各组细胞及其上清。应用实时荧光定量PCR法检测不同组Caspase-1、IL-1β和IL-18 mRNA表达水平的变化；应用ELISA检测细胞上清pro-IL-1β、pro-IL-18、IL-1β和IL-18含量的变化。结果显示，试验成功分离得到致病性大肠杆菌，经PCR检测成功获得HPI irp2基因阳性株，经实时荧光定量PCR法检测后发现HPI⁺组、HPI^-组与空白对照组相比，Caspase-1、IL-1β和IL-18 mRNA表达水平均呈上调趋势，且HPI⁺组高于HPI^-组。ELISA检测结果显示，与空白对照组相比，HPI⁺组和HPI^-组pro-IL-1β、pro-IL-18、IL-1β和IL-18的蛋白表达量普遍呈上调趋势，且HPI⁺组均高于HPI^-组。结果表明，云南撒坝猪致病性大肠杆菌HPI可通过上调猪源巨噬细胞中Caspase-1、IL-1β和IL-18 mRNA的表达量促进猪源巨噬细胞炎性因子IL-1β和IL-18的释放，诱发炎症，最终促进猪源巨噬细胞发生细胞焦亡。

关键词: 致病性大肠杆菌; 高致病性毒力岛; 巨噬细胞; 细胞焦亡

Abstract: In order to study the molecular mechanism of cell death in porcine macrophages induced by pathogenic E.coli HPI from Saba pig in Yunnan,the pig derived macrophages infected with HPI positive strain of Saba pig in Yunnan were taken as the breakthrough point. E.coli was isolated from feces of yellow-white dysentery in a large scale farm in Chuxiong,Yunnan.The HPI irp2 gene was detected by PCR technology.Macrophages were infected with HPI positive strains (HPI⁺) and negative strains (HPI^-),respectively,and LPS+ATP group and blank control group were set up,cells and their supernatants were collected at 0.5 and 6 h.The expression levels of Caspase-1,IL-1β and IL-18 in different groups were detected by Real-time PCR,and the contents of pro-IL-1β,pro-IL-18,IL-1β and IL-18 in cell supernatant were detected by ELISA.The results showed that pathogenic E.coli was successfully isolated and HPI irp2 gene positive strains were successfully obtained by PCR.Real-time PCR results showed that the expression levels of Caspase-1,IL-1β and IL-18 of HPI⁺ and HPI^- groups were all higher than those of blank control group.It showed an upward trend,and HPI⁺ group was higher than HPI^- group.ELISA results showed that compared with blank control group,the expression of pro-IL-1β,pro-IL-18,IL-1β and IL-18 in HPI⁺ and HPI^- groups were generally up-regulated,and the HPI⁺ group was higher than that in HPI^- group.The results showed that the pathogenic E.coli HPI of Saba pig in Yunnan could promote the release of inflammatory cytokines IL-1β and IL-18 from pig macrophages by up regulating the expression of Caspase-1,IL-1β and IL-18 mRNA in porcine macrophages,and eventually induce inflammation.

Key words: pathogenic E.coli; HPI; macrophages; cell death

中图分类号:

S852.61

茶金龙, 刘超英, 高洪, 王世玉, 单春兰, 景麟稀, 王浩, 杨伟. 致病性大肠杆菌HPI对Caspase-1细胞焦亡相关分子表达的影响[J]. 中国畜牧兽医, 2019, 46(11): 3181-3189.

CHA Jinlong, LIU Chaoying, GAO Hong, WANG Shiyu, SHAN Chunlan, JING Linxi, WANG Hao, YANG Wei. Effect of Pathogenic E.coli HPI on the Expression of Burnout-related Molecules in Caspase-1 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3181-3189.

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