致犊牛脑炎大肠杆菌对细胞及小鼠的致病性

Pathogenicity of Escherichia coli causing calf encephalitis to cells and mice

旨在探讨犊牛脑炎大肠杆菌对小鼠脑微血管内皮细胞(BMEC细胞)和小鼠肺泡巨噬细胞(MH-S细胞)以及健康小鼠肺脏、脑组织的炎性损伤机制。研究采用致病性大肠杆菌菌株侵染BMEC细胞和MH-S细胞,观察细胞形态变化;平板计数法检测菌株对细胞的黏附、侵袭能力以及小鼠肺脏、脑组织载菌量;RT-qPCR检测菌株对细胞及小鼠脏器不同时间段的TNF-α、IL-1β、IL-6基因表达量的影响;Western blot检测感染后细胞及小鼠脏器与炎症有关信号通路重要蛋白p-NF-κB、p-JAK2、p-STAT3表达量。结果显示,侵染组细胞培养基浑浊,细胞视野变暗、模糊不清,部分细胞皱缩死亡,产生较多细胞碎片。菌株对BMEC细胞的黏附率和侵袭率3 h显著低于6 h(P<0.050),对MH-S细胞的黏附率和侵袭率在3 h显著高于6 h(P<0.010);感染小鼠脑组织大面积水肿、出血,肺脏有不同程度的肿大和出血,脑、肺脏组织载菌量在12 h最高。与对照组相比,感染组在3和6 h的IL-1β、IL-6、TNF-α的mRNA表达量均显著上升(P<0.050),且BMEC细胞和MH-S细胞的炎性因子mRNA表达量分别在6、3 h最高;感染小鼠脑、肺脏组织的炎性因子mRNA表达量随时间延长呈现先增加后减少的趋势,均在感染12 h有最高的表达量;侵染组的BMEC细胞和MH-S细胞及小鼠肺脏、脑组织的p-NF-κB蛋白表达量显著高于对照组(P<0.001),p-JAK2蛋白和p-STAT3蛋白表达量显著低于对照组(P<0.050)。上述结果表明,致病性大肠杆菌可黏附侵袭BMEC细胞和MH-S细胞,促进细胞及组织NF-κB蛋白表达、抑制JAK2蛋白和STAT3蛋白表达,进而刺激细胞及组织产生炎性反应。

The purpose of this study was to investigate the damage mechanism of pathogenic E.coli on mouse brain microvascular endothelial cells(BMEC cells)and mouse alveolar macrophages(MH-S cells),as well as the lung and brain of healthy mice.In this study,BMEC cells and MH-S cells were infected with pathogenic E.coli strains,and cell morphological changes were observed.Plate counting method was used to detect the adhesion and invasion ability of the strains to cells and the number of bacteria in the lungs and brains of mice.RT-qPCR was used to detect the expression of TNF-α,IL-1βand IL-6 genes in cells and mouse organs at different time periods.Western blot was used to detect the expression of p-NF-κB,p-JAK2 and p-STAT3 proteins related to inflammation in cells and mouse organs after infection.The results showed that the cell culture medium of the infection group was turbid,the cell vision became dark and blurred,some cells shrank and died,and more fragments were produced.The adhesion rate and invasion rate of BMEC cells at 3 h were significantly lower than those at 6 h(P<0.050),and the adhesion rate and invasion rate of MH-S cells at 3 h were significantly higher than those at 6 h(P<0.010).Infected mice had a large area of swelling and bleeding in the brain,and the lungs had different degrees of swelling and bleeding.The bacterial load in the brain and lung was the highest at 12 h.Compared with the control group,the mRNA expression levels of IL-1β,IL-6 and TNF-αin the infection group were significantly increased at 3 h and 6 h(P<0.050),and the mRNA expression levels of inflammatory factors in BMEC cells and MH-S cells were the highest at 6 and 3 h,respectively.The mRNA expression of inflammatory factors in the brain and lung of infected mice showed a trend of increasing first and then decreasing with time,with the highest expression at 12 h after infection.The expression levels of p-NF-κB protein in BMEC cells,MH-S cells,lung and brain tissues of mice in the infection group were significantly higher than those in the control group(P<0.001),and the expression levels of p-JAK2 protein and p-STAT3 protein were significantly lower than those in the control group(P<0.050).The above results showed that pathogenic E.coli could adhere and invade BMEC cells and MH-S cells,colonize in lung and brain tissues of mice,promote the expression of NF-κB protein in cells and tissues,inhibit the expression of JAK2 protein and STAT3 protein,and then stimulate cells and tissues to produce inflammatory response.