›› 2016, Vol. 43 ›› Issue (3): 592-600.doi: 10.16431/j.cnki.1671-7236.2016.03.005

• 生物技术 • 上一篇    下一篇

NF-κB信号通路调控布鲁氏菌胞内存活分子机制的初步研究

易继海1, 张俊波2, 李爽1, 王熙楚1, 张辉1, 陈创夫1   

  1. 1. 石河子大学动物科技学院, 石河子 832000;
    2. 铜仁学院生物与农林工程学院, 铜仁 554300
  • 收稿日期:2015-09-08 出版日期:2016-03-20 发布日期:2016-03-30
  • 通讯作者: 陈创夫 E-mail:ccf-xb@163.com
  • 作者简介:易继海(1989-),男,新疆奎屯人,硕士生,研究方向:家畜病原分子生物学,E-mail:724050645@qq.com
  • 基金资助:
    科技部国家科技重大专项(1104602);国家自然科学基金(31502067)

Preliminary Study on NF-κB Signaling Pathway Regulating Brucella Intracellular Survival Molecular Mechanisms

YI Ji-hai1, ZHANG Jun-bo2, LI Shuang1, WANG Xi-chu1, ZHANG Hui1, CHEN Chuang-fu1   

  1. 1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
    2. College of Biological and Agricultural Engineering, Tongren University, Tongren 554300, China
  • Received:2015-09-08 Online:2016-03-20 Published:2016-03-30

摘要: 本试验用布鲁氏菌强、弱毒株侵染小鼠巨噬细胞RAW264.7,旨在探讨NF-κB信号通路与布鲁氏菌强、弱毒株在胞内生存的关系。采用光滑型牛布鲁氏菌2308、粗糙型牛布鲁氏菌RB51在不同感染复数下侵染小鼠巨噬细胞RAW264.7,侵染0、4、8、24 h后,裂解细胞收集蛋白,Western blotting检测布鲁氏菌对激活NF-κB信号通路的影响。利用不同浓度的NF-κB信号通路抑制剂处理小鼠巨噬细胞RAW264.7,然后用布鲁氏菌在不同感染复数下侵染小鼠巨噬细胞RAW264.7,ELISA试剂盒检测细胞因子TNF-α、IL-1β、IL-6的表达量;同时对胞内菌CFU进行计数。结果显示粗糙型牛布鲁氏菌RB51可以强烈激活NF-κB信号通路,光滑型牛布鲁氏菌2308对其激活作用较弱;同时对NF-κB信号通路的激活具有浓度依赖性,在感染复数为80:1、侵染时间为8 h时光滑型牛布鲁氏菌2308和粗糙型牛布鲁氏菌RB51对NF-κB激活程度最强,且该通路参与产生TNF-α、IL-1β和IL-6;NF-κB信号通路抑制剂BAY11-7082影响布鲁氏菌在胞内的生存。因此,粗糙型牛布鲁氏菌RB51胞内存活与NF-κB信号通路密切相关,为进一步研究布鲁氏菌的胞内致病机制奠定基础,也为布鲁氏菌新型药物的研发、家畜布鲁氏菌病预防和治疗提供科学依据。

关键词: 布鲁氏菌2308; 布鲁氏菌RB51; 巨噬细胞; NF-κB信号通路; 胞内存活

Abstract: By the infection of Brucella virulent strain and attenuated strain in mice macrophage RAW264.7,the assay was aimed to explore the relationship between NF-κB signaling pathways and Brucella virulent strain and attenuated strain in intracellular survival.Use different MOI Brucella (2308,RB51,16M and M5) to infect mice macrophage RAW264.7,after 0,4,8 and 24 h infected,cracking cell and collecting supernatant,we detected the effect of Brucella on activation of NF-κB signaling pathway by Western blotting.Different concentrations of NF-κB signaling pathway inhibitor were incubated with mice macrophage RAW264.7,with different multiplicities of infection (MOI) of Brucella infecting cells,ELISA kits to detect the expressions of TNF-α,IL-1β and IL-6 cytokine;At the same time,count the number of intracellular bacteria of CFU.The results showed that rough cattle Brucella strains RB51 could strongly activate NF-κB signaling pathway,smooth cattle Brucella strains 2308 was weak in the activation;At the same time,the activation of NF-κB signaling pathway was concentration dependent.When the MOI was 80,infection time was 8 h,NF-κB activation degrees of rough cattle Brucella strains RB51 and smooth cattle Brucella strains 2308 were the strongest,and this pathway was involved in producing TNF-α and IL-6;NF-κB signaling pathway inhibitor BAY11-7082 affected Brucella intracellular survival.So rough cattle Brucella strains RB51 intracellular survival and NF-κB signaling pathway activity were closely related.The results laid the foundation for the further study of Brucella intracellular pathogenesis,also provided scientific basis for the research of new drugs to Brucella,and prevention and treatment of brucellosis.

Key words: Brucella 2308; Brucella RB51; macrophages; NF-κB signaling pathway; intracellular survival

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