Establishment of bovine mammary epithelial cells, an in vitro model for milk protein synthesis.

采用胶原酶法分别从妊娠晚期和妊娠早期的荷斯坦奶牛中分离出两种乳腺上皮细胞。(1)从妊娠晚期奶牛（270-d）分离的乳腺上皮细胞。原代培养细胞在10%的FCS DME中，5天内增加4-5倍。细胞污染物，尤其是成纤维细胞，可以被去除，因为在0.1%胰蛋白酶处理后，成纤维细胞比乳腺上皮细胞更早地从胶原包被的培养皿中分离出来。乳腺上皮细胞在基质上形成肺泡状结构，而在i型胶原上只观察到扁平细胞。在低FCS条件下，62 ng/ml剂量的IGF-I刺激细胞增殖。这种效应被同时添加到培养物中的泌乳激素所抑制。(2)早期妊娠奶牛乳腺上皮细胞（26 d）为了克服成纤维细胞生长的问题，采用限制稀释的方法克隆了成纤维细胞。单个细胞产生一个具有上皮形态的集落，细胞角蛋白染色呈阳性。该克隆显示，群体翻倍时间约为24小时，并已培养超过20代没有显示任何衰老的迹象。乳原激素、胰岛素、催乳素和地塞米松对这些细胞增殖的影响在i型胶原包膜培养皿中培养时未观察到。乳原激素在基质上培养，乳腺细胞分化。

We isolated two kinds of mammary epithelial cells by a collagenase method from Holstein cows in late and early pregnancy, respectively.(1) Mammary epithelial cells isolated from a cow in late pregnancy (270-d).Primary cultured cells increased 4-5 times within 5days in 10 % FCS DME on collagen coated dishes. The cellular contaminants, especially fibroblasts, could be removed because fibroblasts detached earlier from collagen-coated dishes than mammary epithelial cells following 0.1 % trypsin treatment Mammary epithelial cells formed an alveolar-like structure on Matrigel, while only flat shaped cells were observed on type-I collagen. IGF-I stimulated the proliferation of the cells at a dose of 62 ng/ml in low FCS condition. This effect was inhibited by concomitant addition of lactogenic hormones to the culture.(2) Mammary epithelial cells from a cow in early pregnancy (26-d)To overcome the problem of fibroblast outgrowth, cells were cloned by limiting dilution. One colony arising from single cell with epithelial morphology and showing positive cytokeratin staining was obtained. The clone showed a population doubling time of approximately 24 h and have been cultured more than 20 passages without showing any sign of senescence. The effect of lactogenic hormones, insulin, prolactin, and dexamethasone, on the proliferation of these cells was not observed when cultured on collagen type-I coated dishes. The mammary cells were differentiated when cultured on Matrigel with lactogenic hormones.