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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：2008 下载量：486
| 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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