胶原酶Ⅰ消化法体外分离培养奶牛乳腺上皮细胞  

Bovine Mammary Epithelial Cells Isolated and Cultured by Collagenase Ⅰ Digestion Method In Vitro

作　　者：徐丹丹[1] 孙志鹏[1] 张旭[1] 刘东宇[1] 许小楠[1] 王乐[1] 陈嘉[1] 王建发[1] 杨彬[1] 武瑞[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319

出　　处：《中国细胞生物学学报》2016年第9期1060-1065,共6页Chinese Journal of Cell Biology

基　　金：黑龙江八一农垦大学研究生创新项目(批准号:YJSCX2015-Y23);国家自然科学基金(批准号:31472249;31402157)资助的课题~~

摘　　要：为获得纯净的奶牛乳腺上皮细胞,该研究采用胶原酶I消化法,在不添加外源激素及生长因子的条件下培养奶牛乳腺上皮细胞。采用差时消化与差速贴壁方法,对奶牛乳腺上皮细胞进行纯化。采用Western blot方法检测细胞中β-酪蛋白(β-casein,CNS2)的水平。通过免疫荧光技术对细胞进行鉴定。结果显示,纯化后的细胞呈现典型的"铺路石"或"鹅卵石"样。细胞角蛋白-18(cytokeratin-18,CK-18)反应呈阳性,波形蛋白反应呈阴性。细胞传至15代冻存并复苏后生长状态依然良好。这提示,胶原酶I消化法在不添加外源激素及生长因子的条件下可以获得奶牛乳腺上皮细胞,以用于后续实验。The aim of this study is to isolate and culture bovine mammary epithelial cells. We used the method of collagenase I digestion to culture bovine mammary epithelial cells without adding exogenous hormones and growth factors. Bovine mammary epithelial cells were purified by different time digestion method and different speed adherence method. The level of β-casein（CNS2） in cells were detected by Western blot. Cells were identified by immunofluorescence. The results showed that the cells had typical cobblestone-like morphology, and positively expressed cytokeratin-18（CK-18）, negative expressed vimentin. The cells of 15 th passage were recovered, and still grown in good condition. This result indicated that collagenase I digestion method can be used to obtain bovine mammary epithelial cells without adding exogenous hormones and growth factors.

关 键 词：胶原酶Ⅰ 奶牛乳腺上皮细胞 原代培养 免疫荧光 

分 类 号：S858.23[农业科学—临床兽医学]