嗜肺军团菌MOMP通过激活NOD2/RIP2信号通路抑制RAW264.7巨噬细胞的吞噬功能并增强其趋化能力  

MOMP of Legionella inhibits phagocytosis of RAW264. 7 macrophages and enhances their chemotaxis by activating NOD2/RIP2 signaling pathway

作　　者：甄庆洁 曹秀琴[2] 卢敬敬 杨志伟[1] ZHEN Qingjie;CAO Xiuqin;LU Jingjing;YANG Zhiwei(Department of Pathogenic Biology and Immunology,School of Basic Medicine,Ningxia Medical University;Ministry-of-Education Key Laboratory for Fertility Preservation and Maintenance,Department of Pathogenic Biology and Immunology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院病原生物学与免疫学系,宁夏银川750004 [2]宁夏医科大学生育力保持省部级共建教育部重点实验室、基础医学院病原生物学与免疫学系,宁夏银川750004

出　　处：《细胞与分子免疫学杂志》2018年第6期488-494,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：西部一流学科-人才培养(30181602); 西部一流学科-开放课题(30181603)

摘　　要：目的探讨嗜肺军团菌主要外膜蛋白(MOMP)对RAW264. 7巨噬细胞吞噬功能和趋化功能的影响并探讨其机制。方法采用MOMP与RAW264. 7巨噬细胞进行体外共培养,用CCK-8法检测MOMP对RAW264. 7巨噬细胞的毒性,确定半数抑制浓度(IC50)。采用(1. 14、0. 57、0. 28)μg/m L MOMP分别处理RAW264. 7巨噬细胞,并设细胞对照组。RAW264. 7巨噬细胞处理24、48、72 h,收集细胞和培养上清,用中性红吞噬实验检测巨噬细胞的吞噬功能;用TranswellTM小室检测巨噬细胞的趋化功能; ELISA检测细胞培养上清单核细胞趋化蛋白1(MCP-1)和白细胞介素10(IL-10)的含量;实时定量PCR检测巨噬细胞核苷酸结合寡聚结构域1(NOD1)、NOD2、受体相互作用蛋白2(RIP2) mRNA水平,Western blot法检测NOD1、NOD2、RIP2的蛋白水平。结果 CCK-8法检测MOMP对RAW264. 7巨噬细胞的IC_(50)为5. 69μg/m L;与对照细胞相比,MOMP处理引起RAW264. 7巨噬细胞吞噬功能降低且呈剂量和时间依赖性;随着MOMP剂量的增加,巨噬细胞的趋化能力及细胞培养上清中MCP-1、IL-10的分泌水平增加,并在36 h达到峰值; NOD2、RIP2的mRNA和蛋白表达水平也增加,NOD2和RIP2的mRNA水平在12 h达到高峰,蛋白水平在24 h达到峰值。结论 MOMP抑制RAW264. 7巨噬细胞的吞噬功能并增强其趋化功能,与激活NOD2/RIP2信号通路有关。Objective To investigate the effect of the main outer membrane protein（ MOMP） of Legionella on the phagocytosis and chemotaxis of RAW264. 7 macrophages and explore its mechanism. Methods MOMP and RAW264. 7 macrophages were cultured in vitro. The toxicity of MOMP to RAW264. 7 macrophages was detected by CCK-8 assay and50% inhibitory concentration（ IC50） was determined. The RAW264. 7 macrophages were treated by MOMP（ 1. 14,0. 57,0. 28） μ/ L and the control group was established. The cells and cultivate supernatants were collected 24,48 and 72 hours after the RAW264. 7 macrophages were treated by MOMP. The phagocytic function of macrophages was detected by the neutral red phagocytosis experiment; the chemotaxis function of macrophage was examined by TranswellTMassay,and the levels of monocyte chemoattractant protein 1（ MCP-1） and interleukin 10（ IL-10） in cell culture supernatant monocytes were detected by ELISA. Real-time quantitative PCR was used to check the mRNA level of macrophage nucleotide-binding oligomerization domain 1（ NOD1）,NOD2 and receptor-interacting protein 2（ RIP2）. The protein levels of NOD1,NOD2 and RIP2 were detected by Western blot analysis. Results The IC_（50） of MOMP on RAW264. 7 macrophages was 5. 69 μ/ L. Compared with the control cells,MOMP treatment caused a decrease of RAW264. 7 macrophage phagocytosis in a dose-and time-dependent manner. With the increase of MOMP dosage,the chemotaxis of macrophages and the secretory levels of MCP-1 and IL-10 in the cell culture supernatant increased,and peaked in 36 hours. The mRNA and protein expression levels of NOD2,RIP2 also increased,mRNA levels of NOD2 and RIP2 peaked in 12 hours,and protein levels peaked in 24 hours.Conclusion MOMP can inhibit the phagocytosis of RAW264. 7 macrophages and enhance its chemotaxis function,which is related to the activation of NOD/IP2 signaling pathway.

关 键 词：嗜肺军团菌 主要外膜蛋白(MOMP) 巨噬细胞 吞噬功能 趋化功能 NOD样受体(NLR) 

分 类 号：R392.12[医药卫生—免疫学] Q256[医药卫生—基础医学]