摘要
目的探讨脂多糖(lipopolysaccharide,LPS)对奶牛乳腺成纤维细胞(bovine mammary fibroblast,BMFB)增殖速率、胞内Toll样受体(Toll-like receptor,TLR)及其信号通路相关基因m RNA表达水平的影响。方法原代培养BMFB,用LPS(终浓度10μg/mL)刺激BMFB不同时间后,经CCK-8法检测BMFB的增殖速率,实时荧光定量PCR法检测BMFB胞内TLR及其信号通路相关分子m RNA的表达水平。结果 LPS刺激BMFB 0、1、3 h后,BMFB增殖速率差异无统计学意义(P>0.05),LPS刺激BMFB 6 h开始,试验组BMFB的增殖速率明显快于对照组(P<0.05)。与对照组比较,LPS刺激BMFB 12 h后,TLR2、TLR4、信号转导通路激活核转录因子κB(nuclear factor of kappa B,NF-κB)、肿瘤坏死因子受体相关因子6(TNF receptor associated factor 6,TRAF6)、白介素-1β(interleukin-1β,IL-1β)、IL-6、IL-8、肿瘤坏死因子-α(tumor necrosis factor alpha-α,TNF-α)和白细胞介素-1受体相关激酶1(interleukin-1 receptor associated kinase 1,IRAK1)基因m RNA表达水平显著升高(P<0.05),髓样分化因子88(myeloid differentiation factor 88,My D88)基因m RNA的表达水平差异无统计学意义(P>0.05)。结论 BMFB可能通过TLR2和TLR4识别LPS,并与NF-κB信号通路级联诱导炎性细胞因子和趋化因子的释放,参与奶牛乳腺先天性免疫应答。
Objective To investigate the effects of lipopolysaccharide（LPS）on proliferation rate of bovine mammary fibroblasts（BMFBs）as well as mRNA expression levels of intracellular Toll-like receptor and its signal pathway-related genes.Methods BMFBs were subjected to primary culture and stimulated with LPS at a final concentration of 10 μg/mL for various hours.The proliferation rate of BMFBs was determined by CCK-8 kit,while the expression levels of Toll-like receptor and its signal pathway-related molecular mRNAs by real-time fluorescent quantitative PCR.Results The proliferation rates of BMFBs showed no significant difference after stimulation with LPS for 0,1 and 3 h（P〈0.05）,while was significantly higher in test group than in control group more than 6 h after stimulation（P〈0.05）.As compared with those in control group,the expression levels of TLR2,TLR4,signal pathway-activated nuclear factor kappa B（NF-κB）,tumor necrosis factor（TNF）receptor associated factor 6（TRAF6）,interleukin-1β（IL-1β）,IL-6,IL-8,TNFα and interleukin-1 receptor associated kinase 1（IRAK1）mRNAs 12 h after stimulation increased significantly（P〈0.05）,while that of myeloid differentiation factor 88（MyD88）mRNA showed no significant change（P〈0.05）.Conclusion BMFB might recognize LPS by TLR2 and TLR4 and cascade with NF-κB signaling pathway to induce the release of inflammatory cytokines and chemokines,and participate in dairy mammary innate immune responses.
作者
张旭
武瑞
王建发
贺显晶
杨彬
徐丹丹
王海
单旭菲
王瑛琪
岳阳
ZHANG Xu;WU Rui;WANG Jian-fa;HE Xian-jing;YANG Bin;XU Dan-dan;WANG Hai;SHAN Xu-fei;WANG Ying-qi;YUE Yang(College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongiiang Province, China)
出处
《中国生物制品学杂志》
CAS
CSCD
2018年第4期362-367,共6页
Chinese Journal of Biologicals
基金
国家自然科学基金项目(31472249
31402157)
黑龙江省普通本科高校青年创新人才培养计划(UNPYSCT-2015087)
黑龙江八一农垦大学博士科研启动基金(XYB2014-12)
黑龙江省自然科学基金面上项目(C2016045)
关键词
奶牛乳腺成纤维细胞
脂多糖
增殖速率
基因表达
Bovine mammary fibroblasts
Lipopolysaccharides （LPS）
Proliferation rate
Gene expression
