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体外奶牛乳腺炎上皮细胞模型的建立

发表时间:2017-05-15  浏览量:1373  下载量:301
全部作者: 张文耀,陈青,和桂良,张涌,高明清
作者单位: 西北农林科技大学动物医学院;西北农林科技大学农业部生物技术重点实验室
摘 要: 目的:体外建立奶牛乳腺炎上皮细胞模型。方法:体外培养奶牛乳腺上皮细胞,用不同浓度(0,l,5,10,20 ng/µL)脂多糖(lipopolysaccharides,LPS)或不同浓度(0,1,10,20,40 ng/µL)脂磷壁酸(lipoteichoic acid,LTA)处理奶牛乳腺上皮细胞,采用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测炎性应答标志肿瘤坏死因子(tumor necrosis factor-α,TNF-α)的基因表达水平,酶联免疫吸附实验(enzymes linked immunosorbent assay,ELISA)检测炎性应答标志TNF-α的蛋白表达水平,进而确定建立模型的LPS和LTA最适处理浓度。然后在确定的最适浓度下,分别以LPS或LTA处理上皮细胞不同时间[LPS(0,3,6 h)或LTA(0,3,6,12,24 h)],采用qRT-PCR检测炎性应答标志TNF-α的基因表达水平,以确定建立模型的最佳处理时间。结果:qRT-PCR和ELISA实验结果显示LPS浓度10 ng/µL处理乳腺上皮细胞3 h可明显诱导TNF-α基因和蛋白表达水平;LTA浓度20 ng/µL处理乳腺上皮细胞12 h明显诱导TNF-α基因和蛋白表达水平。结论:本实验成功地建立了LPS或LTA 诱导体外培养奶牛乳腺上皮细胞的乳腺炎细胞模型。
关 键 词: 兽医学;乳腺炎;脂多糖;脂磷壁酸;乳腺上皮细胞;奶牛
Title: Establishment of a cell model of bovine mastitis in vitro
Author: ZHANG Wenyao, CHEN Qing, HE Guiliang, ZHANG Yong, GAO Mingqing
Organization: College of Veterinary Medicine, Northwest A&F University; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University
Abstract: Objective: To establish an in vitro epithelial cell model of bovine mastitis. Methods: Bovine mammary epithelial cells were routinely cultured in vitro and treated by a series of concentrations (0, 5, 10, 20 ng/µL) lipopolysaccharide (LPS) or a series of concentrations (0, 1, 10, 20, 40 ng/µL) lipoteichoic acid (LTA). The gene expression were analyzed by quantitative real-time PCR (qRT-PCR) and the protein expression were detected by enzymes linked immunosorbent assay (ELISA). The gene and protein expression of TNF-α were used as an indicator to determine the optimal treatment concentration to establish the model. Then, the cells were treated by LPS or LTA for a series of periods (LPS for 0, 3, 6 h; LTA for 0, 3, 6, 12, 24 h) at the optimal concentration. The expression of TNF-α was analyzed by qRT-PCR to determine the optimal processing time. Results: The expression of TNF-α in mammary epithelial cells was significantly upregulated after treated with 10 ng/μL LPS for 3 h or 20 ng/μL LTA for 12 h. Conclusion: An in vitro epithelial cell model of bovine mastitis was successfully established by treating with LPS or LTA.
Key words: veterinary science; mastitis; lipopolysaccharide; lipoteichoic acid; epithelial cells; bovine
发表期数: 2017年5月第9期
引用格式: 张文耀,陈青,和桂良,等. 体外奶牛乳腺炎上皮细胞模型的建立[J]. 中国科技论文在线精品论文,2017,10(9):1037-1042.
 
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