Abstract:

Establishment of mammary gland epithelial cell line for gene targeting is an important step for production of mammary gland bioreactor. Primary bovine mammary gland epithelial cell including a few fibroblast cells were isolated by using Trypsin, Collagenase and their combination. The advantages and shortcomings among different isolation methods were compared. Bovine mammary gland epithelial cells were cultured in vitro by using Trypsin-EDTA stage digestion method. Their cell morphology and growth characteristics were observed when the cell was in the 5th, 10th, 15th and 20th generation to compare the growth effects between cells cultured in single collagen coated dish and multifactor coated dish. The cells epithelial characteristic was identified by immunochemical experiment using cytokeratin-18, which is a specific cytokeratin for mammary gland epithelial cell.

Key words: bovine, mammary gland epithelial cell, in vitro, immunochemical identification

摘要：

建立可用于基因打靶的乳腺上皮细胞系是乳腺生物反应器研制的重要环节,分别采用胰蛋白酶、胶原酶及其两者的混合消化液分离获得了包括少量成纤维细胞在内的原代牛乳腺上皮细胞,比较了不同消化方法的优缺点。利用Trypsin-EDTA阶段消化法在体外培养牛乳腺上皮细胞,观察了此细胞培养到第5代、10代、15代和20代时的细胞形态特征和生长特性,比较了该细胞在胶原基质和混合型基质包被培养皿中的培养效果。以乳腺腺泡上皮细胞中的细胞特征蛋白角蛋白18为标志,利用角蛋白18单克隆抗体进行了细胞免疫组化鉴定,证明分离培养细胞的上皮特性。

关键词: 牛;乳腺上皮细胞系;体外培养;免疫组化鉴定