绵羊肺炎支原体及其脂质相关膜蛋白通过Toll样受体2激活MAPK信号通路诱导小鼠腹腔巨噬细胞TNF-α表达的研究  

Expression of TNF-α in mouse peritoneal macrophages by Mycoplasma ovipneumoniae and LAMPs-treated via Toll-like receptor 2 activation of MAPK signaling pathway

作　　者：白帆 吴金迪 王晓晖 吕天星 王艳芳 郝永清 BAI-Fan;WU Jin-di;WANG Xiao-hui;LYU Tian-xing;WANG Yan-fang;HAO Yong-qing(Laboratory of Microbiology and Immunology,College of Veterinary Medicine,Inner Mongolia Agricultural University,No.306,Zhaowuda Road,Saihan District,Hohhot 010018,China;Veterinary Research Institute,Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences,No.22 Zhaojun Road,Yuquan District,Hohhot 010031,China;Laboratory of Veterinary Pharmacology,and Key Laboratory of Clinical Diagnosis and Treatment Techniques for Animal Disease,Ministry of Agriculture,College of Veterinary Medicine,Inner Mongolia Agricultural University,No.306,Zhaowuda Road,Saihan District,Hohhot 010018,China)

机构地区：[1]内蒙古农业大学兽医学院微生物学与免疫学实验室,内蒙古呼和浩特010018 [2]内蒙古自治区农牧业科学院兽医研究所,内蒙古呼和浩特010031 [3]内蒙古农业大学兽医学院/农业部兽医药理学实验室/动物疫病临床诊疗技术重点实验室,内蒙古呼和浩特010018

出　　处：《中国预防兽医学报》2021年第4期411-417,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：内蒙古农业大学科技成果转化专项资金(YZGC2017027);内蒙古自治区科技计划项目(201802066)。

摘　　要：为验证绵羊肺炎支原体(M. ovi)或脂质相关膜蛋白(LAMPs)是否通过Toll样受体2(TLR2)诱导小鼠腹腔巨噬细胞TNF-α的表达,本研究利用不同浓度的M.ovi或LAMPs刺激C57BL/6J WT小鼠及Tlr2-/-基因缺失小鼠的腹腔巨噬细胞12 h后,利用荧光定量PCR(qPCR)检测Tlr2和TNF-α基因的转录水平。结果显示,C57BL/6J WT小鼠巨噬细胞中Tlr2和TNF-α基因的转录水平均不同程度上升,而Tlr2-/-基因缺失小鼠巨噬细胞TNF-α基因的转录水平明显受到抑制。进一步以终浓度为1×10^(7)cfu/mL M.ovi或8μg/mL LAMPs分别刺激C57BL/6J WT小鼠和Tlr2-/-基因缺失小鼠的巨噬细胞,不同时间后利用qPCR检测Tlr2和TNF-α基因的转录水平;利用流式细胞术检测C57BL/6J WT小鼠巨噬细胞中TLR2蛋白的表达水平;采用ELISA方法检测两种小鼠巨噬细胞上清液中TNF-α的分泌水平;利用western blot检测细胞中MAPK信号通路的激活。qPCR结果显示,以M. ovi或LAMPs经不同时间刺激的C57BL/6J WT小鼠巨噬细胞中Tlr2和TNF-α基因的转录水平均不同程度上升,而Tlr2-/-基因缺失小鼠巨噬细胞中TNF-α基因的转录水平均明显受到抑制;流式细胞术检测结果显示,C57BL/6J WT小鼠巨噬细胞在受到M. ovi或LAMPs刺激后,TLR2蛋白的表达水平均不同程度上升;ELISA结果显示,C57BL/6J WT小鼠巨噬细胞在受到M. ovi或LAMPs刺激后,TNF-α蛋白的表达水平均不同程度上升,而Tlr2-/-基因缺失小鼠巨噬细胞中TLR2蛋白的表达则明显受到抑制;Western blot结果显示,C57BL/6J WT小鼠巨噬细胞经M. ovi或LAMPs刺激后MAPK信号通路中各信号蛋白(Erk、p38、JNK)均发生磷酸化反应,而Tlr2-/-基因缺失小鼠巨噬细胞在受到这两种物质刺激后MAPK信号通路明显受到抑制。上述结果表明,M. ovi及其LAMPs可通过小鼠腹腔巨噬细胞的TLR2激活MAPK信号通路,继而引起细胞因子TNF-α的分泌增多。本研究首次证实M. ovi及其LAMPs是通过TLR2及MAPK信号通路引起小鼠腹To verify whether Mycoplasma ovipneumoniae(M.ovi) or lipid-related membrane proteins(LAMPs) could induce the expression of TNF-α in mouse peritoneal macrophages via Toll-like receptor 2(TLR2), this study used different concentrations of M. ovi or LAMPs to stimulate the peritoneal macrophages from C57 BL/6 J WT mice and Tlr2-/-mice for 12 h. Subsequently, total RNA extracted from the cells was subjected to reverse transcription and fluorescence quantitative PCR(qPCR) to detect Tlr2 and TNF-α gene transcription. The results showed that the transcription levels of Tlr2 and TNF-α genes in C57 BL/6 J WT mouse cells increased with varying degrees, and the transcription levels of TNF-α genes in Tlr2-/-mice were significantly inhibited.Furthermore, the macrophages of C57 BL/6 J WT mice and Tlr2-/-mice were stimulated by a higher concentration of 1×10^(7) cfu/mL M. ovi or 8 μg/mL LAMPs, respectively, and the transcription levels of Tlr2 and TNF-α genes were detected by qPCR after different time periods. The expression level of TLR2 protein in macrophages of C57 BL/6 J WT mice was detected by flow cytometry. The secretion level of TNF-α in the supernatant of macrophages of two kinds of mice was determined by ELISA.Activation of MAPK signaling pathway in cells was detected by western blot. qPCR results showed that the transcription levels of Tlr2 and TNF-α genes in macrophages of C57 BL/6 J WT mice stimulated by M. ovi or LAMPs at different times increased with various degrees, while the transcription levels of TNF-α genes in macrophages of Tlr2-/--mice were significantly inhibited. Flow cytometry results showed that the protein expression of TLR2 in C57 BL/6 J WT mouse macrophages increased to different degrees after being stimulated by M. ovi or LAMPs. ELISA results showed that the expression level of TNF-α protein in macrophages of C57 BL/6 J WT mice increased to different degrees after stimulation by M. ovi or LAMPs, and the expression of TNF-α protein in Tlr2-/-mice was significantly inhibited. This study f

关 键 词：绵羊肺炎支原体 脂质相关膜蛋白 TOLL样受体2 TNF-Α 

分 类 号：S855.1[农业科学—临床兽医学]