猪源粪肠球菌对猪小肠上皮细胞黏附及致炎作用的影响

Adhesion and inflammatory effect of Enterococcus faecalis to intestinal porcine epithelial cells

【目的】通过研究猪源粪肠球菌对肠上皮细胞黏附与致炎作用的影响,探究其致病机制。【方法】利用自行分离鉴定的猪源粪肠球菌N41菌株和N8菌株,通过吉姆萨染色、革兰氏染色、间接免疫荧光检测和场发射扫描电子显微镜观察等手段观察猪源粪肠球菌体外黏附猪小肠上皮细胞(intestinal porcine epithelial cells,IPEC-J2),应用荧光定量PCR方法检测IPEC-J2炎性细胞因子转录水平的变化,以及上皮-间质转化(epithelial-mesenchymal transition,EMT)标志物包括上皮细胞钙黏蛋白(E-cadherin)、神经钙黏蛋白(N-cadherin)、β-连环蛋白(β-catenin)、蜗形蛋白(snail)和波形蛋白(vimen)的转录水平变化。【结果】吉姆萨染色和革兰氏染色镜检结果发现,粪肠球菌能黏附于IPEC-J2,黏附部位位于细胞表面及细胞间紧密连接处,出现聚集现象,呈现不均匀性;携带相同主要毒力基因的粪肠球菌(粪肠球菌N8菌株、粪肠球菌N41菌株)在体外对IPEC-J2的黏附存在差异。荧光定量PCR检测结果显示,感染粪肠球菌后,IPEC-J2中的促炎因子白细胞介素1β(interleukin 1β,IL-1β)、IL-6和IL-8的表达呈现出相似的炎症反应规律,总体趋势为感染后1~4 h炎症反应水平随时间而升高,4~6 h炎症反应降低但仍高于空白对照组细胞。同时,粪肠球菌感染下调了抗炎因子转化生长因子β(transforming growth factorβ,TGF-β)和IL-12的转录水平,以IL-12的下降最显著。此外,不同菌株对于上述细胞因子水平的调节有一定差异,其中N41菌株对促炎因子的上调能力高于N8菌株,而N8菌株对抗炎因子的下调幅度高于N41菌株。EMT标志物检测结果表明,与致病菌肠出血性大肠杆菌O157∶H7相比,猪源粪肠球菌感染IPEC-J2后对EMT标志物的表达水平影响并不明显。【结论】证实了猪源粪肠球菌对IPEC的体外侵袭能力,同时该菌会引起炎性细胞因子的表达升高。

【Objective】This study was conducted to explore the pathogenicity of swine Enterococcus faecalis by observing its adhesion and inflammatory effect to enterocytes in vitro.【Method】Two swine-originated enterococcal isolates N41 and N8 were selected and their adhesion to artificially infected IPEC-J2 cell cultures were observed by means of microscopy after Giemsa’s staining,Gram’s staining,indirect immunofluorescence and field emission scanning electron microscopy as well.Meanwhile,time-line expression changes of several pro-and anti-inflammatory cytokines in the infected IPEC-J2 cells were measured through fluorogenic quantitative PCR examination.The qPCR method was also applied for evaluating the expression of epithelial-mesenchymal transition markers such as E-cadherin,N-cadherin,β-catenin,snail and vimen to explore and compare the effects of Enterococcus faecalis as a conditional pathogen on cell surface marker proteins.【Result】The staining results showed that the E.faecalis bacteria adhered to the IPEC-J2 cells at the cell surface and the intercellular tight junctions,and the bacteria were rather aggregated than evenly distributed.The fluorogenic quantitative PCR data indicated that after infected by the enterococci,the IPEC-J2 cells presented similar responses in the expression of pro-inflammatory cytokines IL-1β,IL-6 and IL-8,elevating at 1 h to 4 h PI(post infection),and decreasing at 4h to 6h PI yet still overcasting those of the control group.Contrarily,down-regulated expression was seen in the anti-inflammatory IL-12 and TGF-βlevels,especially that of IL-12.It was also noticed that there were differences between the two isolates in regulating the cytokine transcription of the infected enterocytes:the N41 bacteria being more potent in up-regulating the pro-inflammatory cytokine levels while the N8 in down-regulating the anti-inflammatory responses.The detection results of EMT markers showed that,compared with the pathogenic E.coli O157∶H7,E.faecalis presented less influence on the expression levels of EMT markers.【Conclusion】It is confirmed that swine E.faecalis possesses the in vitro invasion and inflammatory potentials.