牛溶血性曼氏杆菌对BT细胞炎症因子及其TLR4/NF-κB信号通路影响的研究  

Effects of bovine Mannheimia hemolytica on cytokines and its TLR4/NF-κB signaling pathway in BT cells

作　　者：谢艺萌 刘珊珊[1,2] 苏思雨 蒋鹏轩 张年洁 张泽财 王新[1,2] 朱战波 XIE Yi-meng;LIU Shan-shan;SU Si-yu;JIANG Peng-xuan;ZHANG Nian-jie;ZHANG Ze-cai;WANG Xin;ZHU Zhan-bo(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319,China;Heilongjiang Engineering Research Center of Bovine Disease Prevention and Control,Daqing 163319,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]黑龙江省牛病防控工程技术研究中心,黑龙江大庆163319

出　　处：《中国预防兽医学报》2023年第10期1061-1065,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：“十二五”国家科技支撑计划项目(2014BAD13B03-1);黑龙江省农垦总局“十三五”重点科技计划项目(HNK135-04-03)。

摘　　要：为探究牛溶血性曼氏杆菌(Mh)感染新生牛鼻甲(BT)细胞是否发生炎症反应及其涉及的信号通路,本研究将不同浓度的Mh分别感染BT细胞后经MTT法检测Mh对BT细胞的毒性作用,确定合适的感染浓度。结果显示,与空白对照细胞相比,以终浓度1×10^(4)cfu/mL、1×105cfu/mL的Mh感染后BT细胞的存活率均在80%以上;以浓度1×10^(6)cfu/mL的Mh感染BT细胞后的细胞存活率在60%~70%,以终浓度1×10^(7)cfu/mL的Mh感染BT细胞后的细胞存活率在50%以下;因此确定终浓度1×10^(6)cfu/mL为Mh的最适感染浓度。将Mh以终浓度1×10^(6)cfu/mL感染BT细胞后,采用荧光定量PCR(qPCR)检测细胞中细胞因子(IL-1β、IL-6及TNF-α)m RNA的转录水平;采用间接ELISA检测细胞上清中上述各细胞因子的分泌水平;通过western blot检测BT细胞中TLR4/NF-κB通路中Toll样受体4(TLR4)、p-P65、磷酸化NF-κB抑制蛋白(p-IκBα)的表达水平。q PCR和间接ELISA结果显示,Mh感染的BT细胞中促炎因子IL-6、IL-1β、TNF-αm RNA转录水平及蛋白分泌水平均极显著高于空白对照细胞(P<0.001)。Western blot结果显示,与空白对照组相比,Mh感染的BT细胞中TLR4、p-P65蛋白的表达水平显著升高(P<0.05),p-IκBα蛋白的表达水平极显著降低(P<0.01)。本研究首次表明,Mh感染诱导BT细胞发生了炎症反应,并且该炎症反应是通过激活TLR4/NF-κB信号通路及其相关蛋白的表达实现的。本研究为Mh发病机制的研究及其所致疾病的防治提供参考依据。To investigate whether there is an inflammatory response and the signaling pathways involved in the infection of neonatal bovine nasal turbinate(BT)cells with bovine Mannheimia hemolytica(Mh),the toxic effects of Mh on BT cells were detected by MTT assay after BT cells were infected with different concentrations of Mh,and the appropriate infection concentration was determined.The results showed that compared to the blank control BT cells,the survival rate of BT cells was more than 80%after infection with Mh at 1×10^(4) cfu/mL and 1×105 cfu/mL,the survival rate of BT cells after infection with Mh at 1×10^(6) cfu/mL was 60%-70%,and the survival rate of BT cells infected with Mh at 1×10^(7) cfu/mL was less than 50%.So 1×10^(6) cfu/mL was determined as the optimal infection concentration of Mh.After infected with Mh at 1×10^(6) cfu/mL,the transcription levels of cytokine(IL-1β,IL-6 and TNF-α)mRNA in BT cells were detected by fluorescence quantitative PCR(qPCR),the secretion levels of cytokines in cell supernatants were measured by indirect ELISA,and the expression levels of Toll-like receptor 4(TLR4),p-P65,and phosphorylated NF-κB inhibitory protein(p-IκBα)in TLR4/NF-κB pathway of BT cells were examined by western blot.The results of qPCR and indirect ELISA showed that the mRNA transcription and protein secretion levels of pro-inflammatory cytokines IL-6,IL-1βand TNF-αin Mh-infected BT cells were extremely significantly higher than those in the blank control cells(P<0.001).Western blot results showed that,compared with the blank control group,the expression levels of TLR4 and p-P65 protein significantly increased(P<0.05)and the level of p-IκBαprotein extremely significantly decreased(P<0.01)in Mh-infected BT cells.This research indicated for the first time that Mh infection induces an inflammatory response in BT cells and this response is achieved by activating TLR4/NF-κB signaling pathway and its associated proteins expression.This study provides a reference basis for the study of the pathogenesis o

关 键 词：牛溶血性曼氏杆菌 NF-ΚB信号通路 炎症细胞因子 新生牛鼻甲细胞 

分 类 号：S852.61[农业科学—基础兽医学]