β-防御素-5+mRNA在荷斯坦奶牛乳腺上皮细胞中表达.pdf

摘 要 奶牛乳腺炎是一种复杂的综合性乳腺疾病。它病因复杂，可由多种病原微生物 侵入乳腺而引起，所以防治不易。目前，关于β-防御素的抗乳房炎作用的研究很少。 本研究首先采用胶原酶消化法和胰蛋白酶选择消化法，对荷斯坦奶牛乳腺上皮 细胞进行了分离、培养和纯化，并利用形态学、核型分析和荧光定量RT-PCR检测α- 酪蛋白等方法对所培养乳腺上皮细胞进行了鉴定。随后，通过添加代表大肠杆菌为 主要侵染力的脂多糖 （LPS ）建立了实验性乳房炎的乳腺上皮细胞模型，并采用实时 荧光定量RT-PCR方法检测乳腺上皮细胞中细胞因子IL-6 、IL-12、TNF-α及α-酪蛋白 和β-防御素（BNBD5 ）mRNA 的表达水平。研究结果显示，添加LPS后乳腺上皮细胞 中炎性因子IL-6 、IL-12 和TNF-α 的mRNA 表达量与空白对照组相比显著增加 （p0.01 ），并且α-酪蛋白mRNA表达量显著降低（p0.01 ），说明添加LPS诱发上 皮细胞产生了一定的炎性。并且添加LPS刺激乳腺上皮细胞后，乳腺上皮细胞中 BNBD5 mRNA 的表达量存在着时间和剂量效应，并表现出一定的规律。当脂多糖终 浓度为300ng/mL并培养48h后BNBD5 的表达量最高，与空白对照组间存在极显著差 异（p0.01 ），推测BNBD5基因可能参与了由LPS诱发的奶牛乳房炎的防御机制。本 研究结果将为进一步研究β-防御素的抗乳房炎作用奠定基础。 关键词：乳腺上皮细胞；BNBD5 ；脂多糖；mRNA表达 The Expression of β-Defensin-5 mRNA in Mammary Epithelial Cells of Holstein Cow Abstract Cow Mastitis is a complex and comprehensive breast disease. It can be caused by many pathogenic microorganisms invading mammary gland, so it is difficult to prevent Cow Mastitis. Currently, the researches that using β-defensin to treat mastitis are rare. Firstly, in this study, we used collagenase digestion and trypsin chosen digestion method to isolate and purify bovine mammary epithelial cells. After that, establishing mammary epithelial cells model of experimental mastitis by adding LPS produced mainly by E.coli infection. And real-time PCR was used to detect the levels of mRNA expression of some cytokine in mammary epithelial cells including IL-6, TNF-α, α-casein and BNBD5.The results show that the mRNA expression of inflammatory factor IL-6, IL-12 and TNF-α in mammary epithelial cells were notablely up-regulated (P0.01) compared with the control group and the mRNA expression of α-casein was markedly down-regulated(P0.01), which