金黄色葡萄球菌和表皮葡萄球菌对奶牛中性粒细胞炎性因子分泌的影响

doi:10.16431/j.cnki.1671-7236.2024.08.044

摘要/Abstract

摘要： 【目的】探究金黄色葡萄球菌和表皮葡萄球菌对奶牛外周血中性粒细胞(PMNs)活力以及炎性因子分泌的影响。【方法】以体外培养的奶牛外周血PMNs为研究对象,用姬姆萨和台盼蓝染色对分离培养的奶牛PMNs进行形态鉴定和细胞活率检测,确定奶牛外周血PMNs的最佳作用时间。构建金黄色葡萄球菌、表皮葡萄球菌诱导的奶牛外周血PMNs感染模型,测定奶牛外周血PMNs对两种细菌的杀伤能力;利用CCK-8法测定奶牛PMNs活力,用ELISA法检测细胞中炎性因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-8和IL-10含量。【结果】体外分离培养的奶牛外周血PMNs纯度达99%,其存活率随培养时间延长逐渐降低,24 h内存活率达85%以上。金黄色葡萄球菌和表皮葡萄球菌对数生长期分别为4~7和4~8 h,二者感染奶牛PMNs后,当MOI为10时,2、4、6 h时金黄色葡萄球菌存活率极显著高于表皮葡萄球菌(P＜0.01)。与对照组相比,金黄色葡萄球菌和表皮葡萄球菌感染PMNs不同时间点细胞中TNF-α、IL-8含量均极显著或显著升高(P＜0.01;P＜0.05);感染3、6、12、24 h时细胞中IL-10含量极显著或显著升高(P＜0.01;P<0.05);感染6、9、12、24 h时细胞中IL-1β含量极显著或显著升高(P＜0.01;P＜0.05)。与金黄色葡萄球菌组相比,感染3 h时表皮葡萄球菌组细胞中TNF-α含量极显著降低(P＜0.01),IL-10、IL-8含量极显著或显著升高(P＜0.01;P＜0.05);感染6 h时表皮葡萄球菌组细胞中TNF-α、IL-1β含量极显著降低(P＜0.01);感染9 h时细胞中TNF-α、IL-8含量极显著或显著升高(P＜0.01;P＜0.05)。【结论】金黄色葡萄球菌和表皮葡萄球菌均能逃避奶牛外周血PMNs的吞噬和杀伤作用,二者感染后细胞活力显著降低,能通过调控PMNs中炎性因子的分泌介导先天免疫应答反应,试验结果为靶向PMNs筛选金黄色葡萄球菌性的奶牛乳房炎抗性相关的候选因子研究奠定基础。

关键词: 外周血中性粒细胞; 金黄色葡萄球菌; 表皮葡萄球菌; 炎性因子; 奶牛

Abstract: 【Objective】 The purpose of this experiment was to investigate the effects of Staphylococcus aureus and Staphylococcus epidermidis on the activity of neutrophils (PMNs) and the secretion of inflammatory factors in peripheral blood of dairy cows.【Method】 The morphology and cell viability of PMNs in peripheral blood of dairy cows cultured in vitro were identified by Giemsa and Trypan blue staining,and the optimal time of action of PMNs was determined.The peripheral blood PMNs infection model induced by Staphylococcus aureus and Staphylococcus epidermidis was established to determine the killing ability of peripheral blood PMNs to the two bacteria.PMNs activity was determined by CCK-8 method,and the levels of inflammatory factors tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β),IL-8 and IL-10 were detected by ELISA.【Result】 The results showed that the purity of PMNs from peripheral blood of dairy cows isolated and cultured in vitro reached 99%,and the survival rate decreased gradually with the extension of culture time,and the survival rate reached more than 85% within 24 h.The logarithmic growth periods of Staphylococcus aureus and Staphylococcus epidermidis were 4-7 h and 4-8 h,respectively.When MOI was 10,the survival rate of Staphylococcus aureus at 2,4 and 6 h was extremely significantly higher than that of Staphylococcus epidermidis (P ＜ 0.01).Compared with control group,the contents of TNF-α and IL-8 in PMNs infected by Staphylococcus aureus and Staphylococcus epidermidis were extremely significantly or significantly increased at different time points (P＜0.01 or P＜0.05).The content of IL-10 in cells was extremely significantly or significantly increased at 3,6,12 and 24 h after infection (P＜0.01 or P＜0.05).The content of IL-1β in cells was extremely significantly or significantly increased at 6,9,12 and 24 h after infection (P＜0.01 or P＜0.05).Compared with Staphylococcus aureus group,the content of TNF-α in Staphylococcus epidermidis group at 3 h of infection was extremely significantly decreased (P＜0.01),and the contents of IL-10 and IL-8 were extremely significantly or significantly increased (P＜0.01 or P＜0.05).After 6 h of infection,the contents of TNF-α and IL-1β in Staphylococcus epidermidis group were extremely significantly decreased (P＜0.01).The contents of TNF-α and IL-8 in cells were extremely significantly or significantly increased at 9 h after infection (P＜0.01 or P＜0.05).【Conclusion】 Both Staphylococcus aureus and Staphylococcus epidermidis could evade phagocytic and killing effects of PMNs in peripheral blood of dairy cows, and the cell viability was significantly reduced after infection. Staphylococcus aureus and Staphylococcus epidermidis could mediate the innate immune response by regulating the secretion of inflammatory factors in PMNs of dairy cows.This result laid a foundation for screening candidate factors related to Staphylococcus aureus mastitis resistance by targeting PMNs in dairy cows.

Key words: peripheral blood neutrophils; Staphylococcus aureus; Staphylococcus epidermidis; inflammatory facors; dairy cows

中图分类号:

S852.23

何兴丽, 周佩瑶, 张小雪, 牟泉宙, 李杨, 王昭元, 刘鹏, 王梓, 宋杨阳, 李晓琳, 沈冰蕾. 金黄色葡萄球菌和表皮葡萄球菌对奶牛中性粒细胞炎性因子分泌的影响[J]. 中国畜牧兽医, 2024, 51(8): 3676-3686.

HE Xingli, ZHOU Peiyao, ZHANG Xiaoxue, MOU Quanzhou, LI Yang, WANG Zhaoyuan, LIU Peng, WANG Zi, SONG Yangyang, LI Xiaolin, SHEN Binglei. Effect of Staphylococcus aureus and Staphylococcus epidermidis on the Secretion of Inflammatory Factors by Neutrophils in Dairy Cows[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(8): 3676-3686.

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