生物学杂志

生物学杂志 ›› 2022, Vol. 39 ›› Issue (4): 72-.doi: 10.3969/j.issn.2095-1736.2022.04.072

• 研究报告 • 上一篇    下一篇

干扰TLR4对LPS感染鸡HD11细胞免疫炎症的影响

  

  1. 1. 扬州市职业大学 生物与化工工程学院， 扬州 225012； 2. 扬州大学 动物科学与技术学院，
    扬州225009； 3. 中国教育部国际农业与农产品安全研究联合实验室， 扬州 225009
  • 出版日期:2022-08-18 发布日期:2022-08-15
  • 通讯作者: 孙长花，博士，副教授，主要从事食品与生物技术相关研究，E-mail:echo7260@163.com
  • 作者简介:李欢，博士，讲师，主要从事生物、食品和农业工程相关研究，E-mail: huan.li@me.com
  • 基金资助:
    国家自然科学青年基金项目（31802053）； 江苏省自然科学青年基金项目（BK20180907）； 中国博士后面上基金项目（2019M661950）； 江苏省博士后面上基金项目（137070510）； 江苏省高校“青蓝工程”资助项目； 扬州市第四期“英才培育计划”优秀教育人才资助项目； 扬州市职业大学校级自然科学项目（2018ZR19）

Effect of interference with TLR4 on the immune and inflammation of chicken HD11 macrophages under LPS infection #br#

  1. 1. College of Biological and Chemical Engineering, Yangzhou Polytechnic College, Yangzhou 225012, China;
    2. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; 3. Joint International
    Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou 225009, China
  • Online:2022-08-18 Published:2022-08-15

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摘要： Toll样受体（toll-like receptor, TLR）成员TLR4可识别革兰氏阴性细菌的脂多糖（lipopolysaccharide, LPS），参与宿主免疫炎症反应。研究旨在通过干扰基因TLR4，以阐释TLR4对炎性细胞因子基因表达水平和鸡巨噬细胞HD11凋亡的影响，为完善TLR4在鸡免疫反应中的分子调控机制提供一定依据。不同剂量LPS感染鸡HD11细胞，检测感染后不同时间点TLR4及免疫炎性相关基因表达量。设计3条TLR4基因干扰片段，分别转染鸡HD11细胞，qRT-PCR和流式分别检测LPS感染前后TLR4和免疫炎性相关基因表达量以及细胞凋亡。结果显示，1 μg/mL LPS感染8 h时诱导基因TLR4、IL-1β、IL-8和IL-6的表达效果最佳。成功干扰TLR4基因后，LPS感染鸡HD11细胞时促炎因子基因IL-1β、IL-6、IL-8和TNF-α表达量显著性降低（P<0.05），且有效抑制细胞凋亡；但抗炎因子基因TGF-β和IFN-α的表达水平不受影响（P>0.05）。结果说明，调节TLR4基因的表达可直接影响细胞对LPS的炎症反应，为控制过度炎症反应提供新思路和干预靶点，对缓解应激性病理损伤和提高家禽健康具有实践意义。

关键词: TLR4, LPS, 鸡HD11细胞, 免疫炎症, 敲除载体

Abstract: The toll-like receptors (TLRs) member, TLR4, can recognize a various of lipopolysaccharide (LPS) of gram-negative bacteria to involve host immune and inflammation response. This study was aimed to explore the effects ofTLR4on the expression of inflammatory cytokine genes and chicken macrophage HD11 apoptosis, through interfering the expression ofTLR4, so as to provide some useful information in molecular regulation mechanism ofTLR4on chicken immune response. Different doses of LPS were used to infect chicken HD11 cells, and the expression levels ofTLR4and inflammatory related genes were identified at different time points after infection. TLR4-siRNA were designed and transfected to chicken HD11 cells in vitro, and then quantitative real time PCR (qRT-PCR) and flow cytometry were, respectively, applied to detect the expression of TLR4 and inflammatory related genes, as well as cell apoptosis. The results showed that the optimum condition of LPS was 1 μg/mL infection 8 h to induce the expression of geneTLR4,IL-1β,IL-8andIL-6. In chicken HD11 cells, interferingTLR4gene could significantly decrease the expression of pro-inflammatory genesIL-1β,IL-6,IL-8and TNF-α (P<0.05), and inhibit cell apoptosis under LPS infection, while the expression levels of anti-inflammatory genes TGF-β and IFN-α was decreased with no significant difference (P>0.05). This study confirmed that regulating the expression ofTLR4gene can directly affect the inflammatory response of chicken HD11 cells to LPS, which provide new ideas and intervention targets for controlling excessive inflammatory response, as well as, have practical significance for alleviating stress-induced pathological damage and improving poultry health.

Key words: TLR4, LPS, chicken HD11 macrophages, immune and inflammation, knockdown vector

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