金黄色葡萄球菌对奶牛乳腺成纤维细胞TGF-β1/Smad信号通路及其转分化的影响  

The Effect of Staphylococcusaureuson TGF-β1/Smad Signaling Pathway and Transdifferentiation in Bovine Mammary Fibroblasts

作　　者：杨彬[1] 徐丹丹[1] 孙志鹏[1] 赵佳琦[1] 闫博巍 李晓婷[1] 武瑞[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319

出　　处：《畜牧兽医学报》2016年第7期1495-1501,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：黑龙江八一农垦大学博士后启动基金

摘　　要：为探索金黄色葡萄球菌(S.aureus)对奶牛乳腺成纤维细胞(BMFB)TGF-β1/Smad信号通路及其转分化的影响,用热灭活金黄色葡萄球菌(0、10~4、10~5、10~6、10~7和10~8 CFU·mL^(-1))刺激BMFB,24h后采用Real-time PCR方法检测TGF-β1、α-SMA和collagen-ⅠmRNA的转录量,Western blot方法检测α-SMA、collagen-Ⅰ及p-Smad2/3蛋白的表达量。使用TGF-β1受体特异性抑制剂SB-431542预处理细胞,再用10~5 CFU·mL^(-1)热灭活金黄色葡萄球菌刺激细胞,24h后采用Real-time PCR方法检测α-SMA和collagen-ⅠmRNA的转录量,Western blot方法检测α-SMA、collagen-Ⅰ及p-Smad2/3蛋白的表达量,免疫荧光法检测collagen-Ⅰ蛋白的表达量。结果显示,不同浓度热灭活金黄色葡萄球菌处理细胞的TGF-β1mRNA的转录量显著升高(P<0.05或P<0.01),其中以10~5CFU·mL^(-1)刺激组的TGF-β1mRNA的转录量最高。不同浓度灭活的金黄色葡萄球菌处理细胞的α-SMA、collagen-Ⅰ及p-Smad2/3的表达量均能显著升高(P<0.05或P<0.01),其中以10~5 CFU·mL^(-1)刺激组的α-SMA、collagen-Ⅰ及p-Smad2/3的表达量最高。抑制剂SB-431542能够极显著抑制α-SMA、collagen-Ⅰ及p-Smad2/3的表达量(P<0.01)。结果提示,金黄色葡萄球菌能够通过TGF-β1/Smad信号通路诱导奶牛乳腺成纤维细胞转分化为肌成纤维细胞,本研究为揭示金黄色葡萄球菌性乳腺炎发生硬化的机制奠定基础。This study aimed to investigate the effect of Staphylococcus aureus（S.aureus）on TGF-β1/Smad signaling pathway and transdifferentiation in bovine mammary fibroblasts（BMFB）.BMFB were stimulated with heat-killed S.aureus（HKSA）at 10～4,10～5,10～6,10～7,10～8CFU·mL^（-1）.The transcription of TGF-β1,α-SMAand collagen-Ⅰ mRNA were detected by Realtime PCR after 24 h.The expression ofα-SMA,collagen-Ⅰ and p-Smad2/3protein were detected by Western blot.BMFB were pretreated with TGF-β1receptor-specific inhibitor SB-431542,BMFB were stimulated with heat-killed S.aureus（HKSA）at 10～5 CFU·mL^（-1）.The transcription ofα-SMA and collagen-Ⅰ mRNA were detected by Real-time PCR after 24 h.The expression ofα-SMA,collagen-Ⅰ and p-Smad2/3protein were detected by Western blot.Immunofluorescence was used to detect collagen-Ⅰexpression.The results showed that the transcription level of TGF β1 mRNA,expression ofα-SMA,collagen-Ⅰ and p-Smad2/3increased significantly at different concentration after 24 hstimulation compared with unstimulated BMFB（P 〈0.05）.The expression of TGF-β1,α-SMA,collagen-Ⅰ and p-Smad2/3reached the maximal level at 10～5 CFU·mL^（-1）.The expression ofα-SMA,collagen-Ⅰ and p-Smad2/3were significantly reduced by pretreating BMFB with SB-431542（P 〈0.01）.The results indicate that TGF-β1/Smad signaling pathway plays an important role in S.aureus induced BMFB transdifferentiation.

关 键 词：金黄色葡萄球菌 奶牛乳腺成纤维细胞 α-SMA collagen-Ⅰ P-SMAD2/3 

分 类 号：S852.3[农业科学—基础兽医学]