大肠杆菌对体外培养的奶牛乳腺成纤维细胞TGF-β1和NF-κB及TLR4表达的影响  

Effect of Escherichia coli on TGF-β1,TLR4 and NF-κB mRNA expression and protein secretion in bovine mammary fibroblast cultured in vitro

作　　者：王一[1] 吴建美[1] 毕艳楠[1] 丁玉林[1] 王凤龙[1] 

机构地区：[1]内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018

出　　处：《中国兽医科学》2015年第7期764-770,共7页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31460642)

摘　　要：为研究大肠杆菌对奶牛乳腺成纤维细胞(BMFB)TGF-β1表达的影响以及对其信号转导通路Toll样受体4(TLR4)的表达和核转录因子κB(NF-κB)活化的影响,采用热灭活的不同浓度大肠杆菌菌液(0、1×105、1×106、1×108 CFU/mL)作用于奶牛乳腺成纤维细胞,分别于不同时间点(作用后第6、12、24、48小时)运用实时荧光定量PCR(qRT-PCR)检测TGF-β1和TLR4mRNA的表达,运用Western-blot检测TGF-β1、TLR4和p-NF-κB-p65蛋白的相对表达量。结果显示,大肠杆菌作用BMFB后,TGF-β1mRNA和蛋白的表达量显著的升高,并且TGF-β1mRNA的表达呈明显的时效及量效关系,于第24小时达到高峰后降低(P<0.05);TGF-β1蛋白的表达量呈量效性(P<0.05)。TLR4mRNA和蛋白表达水平显著升高,并且TLR4mRNA和蛋白的表达呈时效性和量效性,分别于作用后第24和12小时达到最高后逐渐下降(P<0.05)。p-NF-κB-p65的表达与大肠杆菌作用浓度呈正量效关系,表达量随作用时间延长先升高后逐渐降低(P<0.05)。上述研究结果表明,热灭活的大肠杆菌能够促进BMFB的TLR4表达,并激活BMFB p-NF-κB-p65蛋白的表达,从而促进TGF-β1的表达。To better understand the effect of heat-inactivated Escherichia coli on the expression of transforming growth factor β1 （TGF-β1）, Toll-like receptor 4 （TLR4） and activation of nuclear transcrip- tion factor κB（NF-κB） in bovine mammary fibroblasts（BMFB） ,BMFBs were cultured in vitro and exposed to the heat-inactivated E. coli at 1 × 10^5 CFU/mL,12× 10^6 CFU/mL,and 1 × 10^8 CFU/mL,respectively,and the total RNA and protein were extracted from the cells at different time（at hour 6,12,24 and 48 post- exposure）, the mRNA expressions of TGF-β1 and TLR4 were examined by real-time RT-PCR and the pro- tein expressions of TGF-β1,TLR4 and p-NF-κB-p65 were analyzed by Western-blot. In result, mRNA and protein expression of TGF-β1 were significantly increased after BMFB treatment and the mRNA expressions of TGF-β1 were in a time and dose-dependent manner. The expression of TGF-β1 peaked at 24 h post-exposure（P 〈0.05）. The increase in TGF-β1 expression was in a time and dose-dependent manner, and peaked at 24 h post-ex- posure（P 〈 0. 05）. And the TGF-β1 secretion was in a dose-dependent manner （P 〈 0. 05）. The mRNA and protein expression of TLR4 was in time and dose-dependent manner,the mRNA peaked at 24 h post- exposure and the protein peaked at 12 h post-exposure（P〈0.05）. And the expression of p-NF-κB-p65 was in dose-dependent manner（P〈0.05）. The results suggested that heatqnactivated E. coli up-regulated the TLR4 expression,activated NF-κB and induced the expression of TGF-β1 in BMFB.

关 键 词：大肠杆菌 奶牛乳腺成纤维细胞 TGF-Β1 TLR4 NF-ΚB 

分 类 号：S852.612[农业科学—基础兽医学]