SIGIRR对奶牛乳腺上皮细胞TLR4致炎信号通路的负调控作用  

SIGIRR attenuates Toll-like receptor 4-mediated immune response in bovine mammary epithelial cells

作　　者：周秋格 刘畅[1] 赖金伦 郭爱珍[1,2] 胡长敏 

机构地区：[1]华中农业大学动物医学院,湖北武汉430070 [2]华中农业大学农业微生物学国家重点实验室,湖北武汉450070

出　　处：《中国兽医学报》2018年第2期404-410,共7页Chinese Journal of Veterinary Science

基　　金：国家肉牛/牦牛产业技术体系资助项目(nycytx-38);宁夏肉牛呼吸疾病综合征及皮肤病防控技术研究与示范资助项目(2012ZDN0903);中央高校基本科研业务费专项资金资助项目(2662015PY054);湖北省自然科学基金资助项目(2015CFB435)

摘　　要：旨在探究SIGIRR对奶牛乳腺上皮细胞（BMECs）TLR4信号通路的负调控作用。构建SIGIRR—pEGFP真核表达载体，脂质体法转染HEK293T及BMECs，荧光显微镜观察及Westernblot验证融合蛋白SIGIRR—pEGFP的表达；LPS刺激转染后BMECs，检测TLR4及其信号通路分子IRAK1、IRAK4和TRAF6的变化，以及下游炎性因子IL-6、IL-8、IL-18和TNF-a的表达量。结果显示，成功构建SIGIRR-pEGFP真核表达载体，荧光检测重组质粒转染到HEK293T和BMECs中；Westernblot鉴定融合蛋白SIGIRR—pEGFP表达正确；重组质粒转染BMECs后，SIGIRR表达量较转染空载质粒组及空白未转染组显著升高（P〈0．01）；炎性因子IL-6、IL-8、IL-10、TNFa、TLR4及下游信号分子IRAK1、IRAK4、TRAF6表达量显著降低（P〈0．01），转染空载质粒组与空白未转染组相比仅IRAKl表达量有差异（P〈0．05）。结果表明，本试验通过研究牛SIGIRR在BMECs中的作用，证实sIGIRR基因具有炎性负调控LPS-TLR4信号通路的作用，具有潜力成为临床治疗奶牛乳腺炎的新靶标。Bovine mastitis is a critical clinic disease that affects the healthy development of milk cow industry. We focused on the negatively modulate effect of SIGIRR on LPS-TLR4 singal pathway in BMECs. The recombinant plasmid SIGIRR-pEGFP was constructed, then HEK293T cells and BMECs were transfected with SIGIRR-pEGFP by Lipofectamine2000 transfection reagent,respec- tively. The expression of SIGIRR-pEGFP was conformed by fluorescence microscope and Western blot analysis. The expression of TLR4 and signal molecules including IRAK1 ,IRAK4,TRAF6,its downstream inflammatory factor such as IL-6, IL 8, IL-I~ and TNF-a was assessed by real-time quantitative RT-PCR after BMECs were stimulated with LPS. The recombinant plasmid SIGIRR- pEGFP was constructed correctly,and it could express through fluorescence microscope and West- ern blot analysis in HEK293T cell and BMECs. The expression of SIGIRR gene increased signifi- cantly compared with empty vector and black control group （P〈0.01）. The expression of TLR4, IRAK 1, IRAK4, TRAF6, its downstream inflammatory factor such as IL-6, IL-8, IL-1β, TNF-α was decreased significantly （P〈0.01）. No significant difference except the IRAK1 gene was found be- tween empty vector group and the untreated BMECs group. In summary, this study firstly ex- plored the function of SIGIRR gene in BMECs and the results verify that SIGIRR gene can attenuate the LPS-TLR4 singal pathway,which provide evidence that SIGIRR gene could be taken as a drug target for clinical treatment of bovine mastitis.

关 键 词：奶牛 乳腺炎 乳腺上皮细胞 SIGIRR TLR4信号通路 负性调控 

分 类 号：S852.2[农业科学—基础兽医学] S857.26[农业科学—兽医学]