大肠杆菌对奶牛子宫内膜上皮细胞炎性损伤的研究

doi:10.16431/j.cnki.1671-7236.2021.07.039

摘要/Abstract

摘要： 试验旨在研究大肠杆菌（E.coli）对奶牛子宫内膜上皮细胞（bovine endometrial epithelial cells，BEECs）的体外炎性损伤，探究大肠杆菌引发炎性反应的最佳浓度、作用时间及机制。首先，用不同浓度的大肠杆菌（5×10⁴、5×10⁵、1×10⁶、2.5×10⁶、5×10⁶ CFU/mL）诱导刺激细胞3、6和9 h，通过倒置显微镜观察细胞形态、CCK-8法测D_{450 nm}值，检测大肠杆菌对细胞活性的影响；其次，用不同浓度的大肠杆菌（5×10⁴、5×10⁵ CFU/mL）处理细胞3、6和9 h，用ELISA方法检测细胞上清液中白介素-1β（IL-1β）、IL-6、IL-8和肿瘤坏死因子-α（TNF-α）的分泌量；最后，用不同浓度的大肠杆菌（5×10⁴、5×10⁵ CFU/mL）处理细胞6和9 h，用Western blotting检测核因子κB抑制蛋白α（IκBα）和p65蛋白的磷酸化水平。结果显示，与对照组相比，大肠杆菌感染细胞9 h后，1×10⁶、2.5×10⁶和5×10⁶ CFU/mL大肠杆菌组细胞活性均极显著降低（P<0.01），5×10⁵ CFU/mL大肠杆菌组显著降低（P<0.05）；大肠杆菌感染细胞9 h后，5×10⁵ CFU/mL大肠杆菌组IL-1β、IL-6、IL-8和TNF-α极显著升高（P<0.01）；大肠杆菌感染细胞6 h后，5×10⁵ CFU/mL大肠杆菌组IκBα、p65蛋白磷酸化水平和IL-6均极显著升高（P<0.01），5×10⁴ CFU/mL大肠杆菌组IκBα和p65蛋白磷酸化水平显著升高（P<0.05）。结果表明，大肠杆菌可以刺激奶牛子宫内膜上皮细胞产生炎性反应，且当细胞与5×10⁵ CFU/mL大肠杆菌作用6 h或与5×10⁴ CFU/mL大肠杆菌作用9 h为最佳。

关键词: 奶牛子宫内膜上皮细胞; 大肠杆菌; 炎性反应; 细胞因子

Abstract: The purpose of this experiment was to study the in vitro inflammatory damage of bovine endometrial epithelial cells caused by Escherichia coli (E.coli),and explore the optimal concentration and duration time of E.coli to induce inflammatory reactions and its mechanism.Firstly,different concentrations of E.coli (5×10⁴,5×10⁵,1×10⁶,2.5×10⁶,5×10⁶ CFU/mL) were used to infect the cells for 3,6 and 9 h,then detected the effect of E.coli on cell viability by observing the cells morphology with inverted microscope morphology and measured the D_{450 nm} with CCK-8 method;Secondly,different concentrations of E.coli (5×10⁴,5×10⁵ CFU/mL) were used to infect the cells for 3,6 and 9 h,then detected the secretion levels of IL-1β,IL-6,IL-8 and TNF-α in the cell supernatant with ELISA method.Finally,different concentrations of E.coli (5×10⁴,5×10⁵ CFU/mL) were used to infect the cells for 6 and 9 h,then the phosphorylation level of IκBα and p65 protein were detected by Western blotting.The results showed that compared with control group,the cell viability of 1×10⁶,2.5×10⁶ and 5×10⁶ CFU/mL E.coli groups was extremely significantly reduced after 9 h of E.coli infection (P<0.01),while the cell viability of 5×10⁵ CFU/mL E.coli group was significantly decreased (P<0.05).The levels of IL-1β,IL-6,IL-8 and TNF-α of 5×10⁵ CFU/mL E.coli group were extremely significantly increassed after 9 h infection (P<0.01).Moreover,the levels of IκBα and p65 phosphorylation protein were significantly higher than those in both 5×10⁴ CFU/mL and control groups (P<0.01).The levels of IL-6,IκBα and p65 phosphorylation protein in 5×10⁵ CFU/mL E.coli group were extremely significantly increased after 6 h infection (P<0.01),while IκBα and p65 phosphorylation protein in the 5×10⁴ CFU/mL E.coli group were significantly increased after 6 h infection (P<0.05).In conclusion,bovine endometrial epithelial cells inflammatory response could be induced by E.coli and the optimal action time was 6 h when E.coli infection concentration was 5×10⁵ CFU/mL,and 9 h when E.coli infection concentration was 5×10⁴ CFU/mL.

Key words: bovine endometrial epithelial cells; Escherichia coli; inflammation; cytokines

中图分类号:

S823.9⁺1

李博通, 王仕宇, 刘佳玮, 张玮琪, 倪耀娣, 刘明超. 大肠杆菌对奶牛子宫内膜上皮细胞炎性损伤的研究[J]. 中国畜牧兽医, 2021, 48(7): 2627-2634.

LI Botong, WANG Shiyu, LIU Jiawei, ZHANG Weiqi, NI Yaodi, LIU Mingchao. Effect of Escherichia coli on Inflammatory Damage of Bovine Endometrial Epithelial Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2627-2634.

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