Singh K, Davis S R, Dobson J M, Molenaar A J, Wheeler T T, Prosser C G, Farr V C, Oden K, Swanson K M, Phyn C V C, Hyndman D L, Wilson T, Henderson H V, Stelwagen K
AgResearch Ltd., Ruakura Research Centre, PB 3123, Hamilton, New Zealand.
J Dairy Sci. 2008 Jun;91(6):2236-46. doi: 10.3168/jds.2007-0900.
We have used cDNA microarray analysis to identify genes that play a role in bovine mammary involution. Involution was induced by termination of milking, and alveolar tissue was collected from 48 nonpregnant Friesian cows in mid lactation sacrificed at 0, 6, 12, 18, 24, 36, 72, and 192 h (n = 6/group) postmilking. The most highly upregulated genes were those associated with oxidative stress. Quantitative real-time reverse-transcription PCR analysis confirmed that mRNA expression of spermidine/spermine N(1)-acetyltransferase was increased by 24 h, superoxide dismutase 2 and metallothionein 1A by 36 h, and glutathione peroxidase by 72 h postmilking. The mRNA expression of the host defense proteins lactoferrin and lingual antimicrobial peptide were increased by 192 h postmilking. A dramatic increase in the protein expression of lactoferrin by 192 h postmilking was also detected by Western analysis. Decreased mRNA expression of the milk protein genes alpha(S1)-, beta-, and kappa-casein, and alpha-lactalbumin were early events in the process of involution occurring within 24 to 36 h postmilking, whereas beta-lactoglobulin mRNA was decreased by 192 h postmilking. Decreases in alpha-lactalbumin and beta-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192 h postmilking, respectively, and the cell survival factors beta1-integrin and focal adhesion kinase were decreased by 72 and 192 h postmilking, respectively. The results demonstrate that in the bovine mammary gland, decreased milk protein gene expression and cell survival signaling are associated with multiple protective responses to oxidative stress that occur before the induction of immune responses and mammary epithelial cell apoptosis during involution.
我们利用cDNA微阵列分析来鉴定在奶牛乳腺退化过程中发挥作用的基因。通过停止挤奶诱导退化，在挤奶后0、6、12、18、24、36、72和192小时（每组n = 6）处死48头处于泌乳中期的非妊娠弗里斯兰奶牛，并收集腺泡组织。上调程度最高的基因是那些与氧化应激相关的基因。定量实时逆转录PCR分析证实，精胺/精胺N(1)-乙酰转移酶的mRNA表达在挤奶后24小时增加，超氧化物歧化酶2和金属硫蛋白1A在挤奶后36小时增加，谷胱甘肽过氧化物酶在挤奶后72小时增加。宿主防御蛋白乳铁蛋白和舌抗菌肽的mRNA表达在挤奶后192小时增加。蛋白质印迹分析也检测到挤奶后192小时乳铁蛋白的蛋白质表达急剧增加。乳蛋白基因α(S1)-、β-和κ-酪蛋白以及α-乳白蛋白的mRNA表达降低是退化过程中的早期事件，发生在挤奶后24至36小时内，而β-乳球蛋白mRNA在挤奶后192小时降低。腺泡组织中α-乳白蛋白和β-乳球蛋白的蛋白质水平分别在挤奶后24小时和192小时降低，细胞存活因子β1-整合素和粘着斑激酶分别在挤奶后72小时和192小时降低。结果表明，在奶牛乳腺中，乳蛋白基因表达降低和细胞存活信号传导与退化过程中免疫反应和乳腺上皮细胞凋亡诱导之前发生的多种氧化应激保护反应相关。
