金黄色葡萄球菌体外刺激奶牛外周血中性粒细胞对炎性因子分泌的影响  

Effects of S.aureus Stimulation in Vitro on Secretions of Inflammatory Cytokines in Peripheral Blood Neutrophils of Dairy Cows

作　　者：张凯 姜雪莹 钱英红 曹金山[1,2] 刘博 张静[1,2] 毛伟 ZHANG Kai;JIANG Xue-ying;QIAN Ying-hong;CAO Jin-shan;LIU Bo;ZHANG Jing;MAO Wei(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot,Inner Mongolia,010018,China;Key Laboratory of Clinical Diagnosis and Treatment Technology of Animal Diseases,Ministry of Agriculture and Rural Affairs,Hohhot,Inner Mongolia,010018,China;Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot,Inner Mongolia,010018,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]农业农村部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018 [3]内蒙古自治区农牧业科学院,内蒙古呼和浩特010018

出　　处：《动物医学进展》2022年第8期30-34,共5页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31860720);内蒙古自治区科学计划项目(2020GG0042);内蒙古自治区自然科学基金项目(2019MS03053)。

摘　　要：以体外培养的奶牛中性粒细胞为研究对象,用瑞氏姬姆萨染色和台盼蓝拒染法对分离培养的奶牛外周血中性粒细胞进行鉴定和活性检测,ELISA检测金黄色葡萄球菌感染体外培养的奶牛外周血中性粒细胞IL-1β、TNF-α、IL-10及IL-8的分泌情况,Western blot检测金黄色葡萄球菌感染体外培养的奶牛外周血中性粒细胞中NF-κB的激活效应。结果显示,体外分离培养的奶牛外周血中性粒细胞纯度达99%,随着培养时间增加奶牛外周血中性粒细胞活性逐渐降低。ELISA检测结果显示,在3 h~24 h刺激时间范围内,随着金黄色葡萄球菌刺激时间的增加,与空白对照组相比体外培养的奶牛外周血中性粒细胞中IL-10、TNF-α、IL-1β和IL-8的分泌量均显著升高(P<0.001)。Western blot检测结果显示,在15 min~120 min刺激时间范围内,随着SA113感染时间的增加,与空白对照组相比体外培养的奶牛外周血中性粒细胞中IκBα磷酸化水平显著升高(P<0.001)。研究结果表明,成功分离培养了奶牛外周血中性粒细胞;金黄色葡萄球菌(SA113株)刺激体外分离培养的奶牛外周血中性粒细胞过程中细胞因子IL-10、TNF-α、IL-1β和IL-8的分泌量随着刺激时间的增加逐渐升高,这一促进作用可能与NF-κB的激活有关,在金黄色葡萄球菌感染奶牛外周血中性粒细胞引发的炎症反应过程中产生调控作用。In this study, the in vitro cultured cow neutrophils were used for the identification and activity assay of isolated cultured cow peripheral blood neutrophils by using Richter’s Kimsa staining and Trypan Blue rejection method.ELISA was used for detecting the secretions of IL-1β,TNF-α,IL-10 and IL-8 from peripheral blood neutrophils of SA113-infected cows cultured in vitro.Western blot was used for detecting the activating effect of NF-κB in peripheral blood neutrophils from SA113-infected cows cultured in vitro.The results showed that the purity of peripheral blood neutrophils from cows isolated and cultured in vitro reached 99%,and the activity of peripheral blood neutrophils from cows decreased gradually with increasing culture time.ELISA results showed that the secretions of IL-10,TNF-α,IL-1β and IL-8 in peripheral blood neutrophils from cows cultured in vitro was significantly higher(P<0.001) with increasing SA113 stimulation time in the range of 3 to 24 h compared with the blank control.Western blot results showed that the level of IκBα phosphorylation in peripheral blood neutrophils of cows cultured in vitro was significantly higher(P<0.001) with increasing time of SA113 infection compared to blank control in the stimulation time range of 15-120 min.The results of the study showed that peripheral blood neutrophils from cows were successfully isolated and cultured.The secretions of cytokines IL-10,TNF-α,IL-1β and IL-8 during the stimulation of S.aureus(strain SA113) in cows’ peripheral blood neutrophils isolated and cultured in vitro gradually increased with the increase of stimulation time, and this promotion effect may be related to the activation of NF-κB,which plays a regulatory role in the inflammatory response triggered by S.aureus infection of cows’ peripheral blood neutrophils.This may be related to the activation of NF-κB,which has a regulatory role in the inflammatory response triggered by S.aureus infection of peripheral blood neutrophils in cows.

关 键 词：金黄色葡萄球菌 外周血中性粒细胞 核因子-ΚB 细胞因子 奶牛 

分 类 号：S852.611[农业科学—基础兽医学]