Immunorelevant gene expression in LPS-challenged bovine mammary epithelial cells.
Immunorelevant gene expression in LPS-challenged bovine mammary epithelial cells.
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DOI：
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发表时间：
2005
影响因子：
2.4
通讯作者：
R. Pareek;O. Wellnitz;R. van Dorp;J. Burton;D. Kerr
中科院分区：
文献类型：
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作者：
R. Pareek;O. Wellnitz;R. van Dorp;J. Burton;D. Kerr
牛乳腺的感染除了引起动物痛苦之外，还是乳制品工业的主要经济负担。更好地了解宿主对感染的初始反应可能会导致更准确地选择耐药动物或新的预防或治疗干预策略。上皮细胞通过向免疫系统发出感染警报并提供感染所在位置的信号，在宿主反应中发挥作用。为了更好地理解这一过程，使用cDNA微阵列方法来搜索乳腺上皮细胞在暴露于大肠杆菌脂多糖（LPS）时产生的潜在信号。从4荷斯坦奶牛的上皮细胞的单独培养物的总RNA收获后6小时LPS的挑战或控制条件。对于每头奶牛，将来自对照或LPS暴露细胞的RNA转录为cDNA并用Cy 3或Cy 5标记，然后合并并应用于含有1278个独特转录物的牛总白细胞（BOTL）微阵列载玻片。使用染料反转，使得来自两种对照培养物的RNA用Cy 3标记，而来自另外两种对照培养物的RNA用Cy 5标记。从得到的微阵列数据中，我们选择了9个基因中的4个显著（P 1.25倍）响应于LPS暴露用于更详细的分析。其中RANTES/CCL 5、IL-6和T-PA 3个基因的阵列信号强度相对较低，但定量实时RT-PCR（Q-RT-PCR）分析显示它们分别被诱导了208倍、10倍和3倍。通过微阵列分析显示最大倍数诱导（2.5倍）的基因是CXCL 5。该基因在阵列上具有相对强的信号强度，并且通过北方印迹分析容易地检测到，其指示10倍诱导。该细胞培养模型系统为乳腺上皮细胞在启动对感染的先天性应答中的重要作用提供了证据。
Infection of the bovine mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Greater understanding of the initial host response to infection may lead to more accurate selection of resistant animals or to novel prophylactic or therapeutic intervention strategies. The epithelial cell plays a role in the host response by alerting the immune system to the infection and providing a signal as to where the infection is located. To understand this process better, a cDNA microarray approach was used to search for potential signals produced by mammary epithelial cells in response to exposure to Escherichia coli lipopolysaccharide (LPS). Total RNA from separate cultures of epithelial cells from 4 Holstein cows was harvested 6 h after LPS challenge or control conditions. For each cow, RNA from control or LPS-exposed cells was transcribed to cDNA and labeled with Cy3 or Cy5, then pooled and applied to a bovine total leukocyte (BOTL) microarray slide containing 1278 unique transcripts. Dye reversal was used so that RNA from two of the control cultures was labeled with Cy3 while RNA from the other two control cultures was labeled with Cy5. From the resulting microarray data we selected 4 of the 9 genes significantly (P 1.25-fold) in response to LPS exposure for more detailed analysis. The array signal intensity for 3 of these genes, RANTES/CCL5, IL-6 and T-PA, was relatively low, but quantitative real-time RT-PCR (Q-RT-PCR) analysis revealed that they were induced 208-fold, 10-fold and 3-fold, respectively. The gene that showed the greatest fold induction by microarray analysis (2.5-fold) was CXCL5. This gene had a relatively strong signal intensity on the array and was easily detected by northern blot analysis, which indicated a 10-fold induction. This cell culture model system provides evidence for an important role of the mammary epithelial cell in initiating the innate response to infection.