Physiology Weihenstephan, Technische Universität München, Freising-Weihenstephan, Germany.
J Anim Physiol Anim Nutr (Berl). 2013 Aug;97(4):647-55. doi: 10.1111/j.1439-0396.2012.01305.x. Epub 2012 Apr 28.
A dietary energy restriction to 49% of total energy requirements was conducted with Red Holstein cows for three weeks in mid-lactation. At the last day of the restriction phase, primary bovine mammary epithelial cells (pbMEC) of eight restriction (RF) and seven control-fed (CF) cows were extracted out of one litre of milk and cultured. In their third passage, an immune challenge with the most prevalent, heat-inactivated mastitis pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was conducted. Lactoferrin (LF) was determined on gene expression and protein level. An enzyme-linked immunosorbent assay (ELISA) was developed to determine LF in milk samples taken twice weekly throughout the animal trial, beginning on day 20 pp (post-partum) until day 150 pp, in cell culture total protein and in cell culture supernatant. Milk LF increased throughout the lactation and decreased significantly during the induced energy deficiency in the RF group. At the beginning of realimentation, LF concentration increased immediately in the RF group and reached higher levels than before the induced deficit following the upward trend seen in the CF group. Cell culture data revealed higher levels (up to sevenfold up-regulation in gene expression) and significant higher LF protein concentration in the RF compared to the CF group cells. A further emphasized effect was found in E. coli compared to S. aureus exposed cells. The general elevated LF levels in the RF pbMEC group and the further increase owing to the immune challenge indicate an unexpected memory ability of milk-extracted mammary cells that were transposed into in vitro conditions and even displayed in the third passage of cultivation. The study confirms the suitability of the non-invasive milk-extracted pbMEC culture model to monitor the influence of feeding experiments on immunological situations in vivo.
在泌乳中期，对红荷斯坦奶牛进行了为期 3 周的总能量需求 49%的饮食能量限制。在限制阶段的最后一天，从 8 头限制（RF）和 7 头对照（CF）奶牛的 1 升牛奶中提取了原代牛乳腺上皮细胞（pbMEC）并进行培养。在第 3 次传代时，用最常见的巴氏消毒乳腺炎病原体大肠杆菌（E. coli）和金黄色葡萄球菌（S. aureus）对其进行了免疫挑战。测定乳铁蛋白（LF）的基因表达和蛋白水平。开发了酶联免疫吸附试验（ELISA），以确定在动物试验期间每周两次采集的奶样中的 LF，从产后第 20 天（pp）开始，直到第 150 天 pp，细胞培养总蛋白和细胞培养上清液。LF 在泌乳期间增加，并在 RF 组诱导能量不足期间显著下降。在重新喂养开始时，RF 组的 LF 浓度立即增加，并在 CF 组的上升趋势之后，达到了诱导性缺陷前的更高水平。细胞培养数据显示，RF 组的细胞中 LF 水平更高（基因表达上调高达 7 倍），且 LF 蛋白浓度显著更高。与暴露于 S. aureus 的细胞相比，在暴露于 E. coli 的细胞中发现了进一步强调的作用。RF pbMEC 组中普遍升高的 LF 水平以及由于免疫挑战而进一步增加的 LF 水平表明，从乳腺中提取的乳腺细胞具有出乎意料的记忆能力，即使在体外培养的第 3 次传代中也能显示出来。该研究证实了非侵入性牛奶提取 pbMEC 培养模型适合监测喂养实验对体内免疫情况的影响。
