奶牛乳腺上皮细胞系的培养与鉴定  

Culture and Identification of the Bovine Mammary Epithelial Cell Line

作　　者：詹康[1] 贡笑笑 左晓昕 陈银银[1] 占今舜[1] 赵国琦[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《动物营养学报》2015年第8期2544-2550,共7页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：江苏省高校优势学科建设工作资助项目(PAPD)

摘　　要：本试验旨在培养功能性奶牛乳腺上皮细胞系,为奶牛泌乳调控和奶牛乳房炎发病机制研究提供功能性的细胞模型。采用组织块细胞培养法来分离纯化并鉴定奶牛乳腺上皮细胞;采用有限稀释法单克隆奶牛乳腺上皮细胞;采用噻唑蓝（MTT）法来分析奶牛乳腺上皮细胞的生长曲线是否为正常的＂S＂形;观察细胞角蛋白18免疫荧光来证明所培养的细胞为上皮型;选择培养至10和20代的奶牛乳腺上皮细胞进行染色体核型分析。结果表明：1）利用组织块细胞分离法能够成功获得奶牛乳腺上皮细胞并传至20代。2）培养5~6 d成纤维细胞迅速增殖且周围分裂出少量的上皮细胞。培养8 d奶牛乳腺上皮细胞迅速增殖,形成岛屿状集落,呈单层＂鹅卵石＂和＂铺路石＂形态生长。3）奶牛乳腺上皮细胞角蛋白18鉴定为阳性。4）培养至10和20代的奶牛乳腺上皮细胞染色体数为60条,具有正常的细胞二倍体核型。综上所述,采用组织块培养细胞能够获得具有稳定性、功能性的奶牛乳腺上皮细胞,但不是永生化细胞。To establish a functional model of bovine mammary epithelial cell line for the study of lactation regulation,pathogenesis of mastitis,this research applied tissue culture to isolate and culture bovine mammary epithelial cells; the limiting dilution method to purify bovine mammary epithelial cells; methyl thiazolyl tetrazolium（MTT） method was used to identify whether the growth curve of bovine mammary epithelial cells was‘S ＇type or not; cytokeratin 18 immunofluorescence was observed to prove that the cells were epithelial type; karyotype analysis was carried out on bovine mammary epithelial cells in passages 10 and 20.The results showed as follows： 1） bovine mammary epithelial cells could be successfully cultured and passaged 20 generations by tissue culture method.2） At 5 to 6 days of cultivation,the fibroblast cells proliferated rapidly and a small amount of epithelial cells divided.At 8 days of cultivation,bovine mammary epithelial cells proliferated rapidly and formed island-like colonies,and grewlike ‘cobblestone＇and ‘slabstone＇as monolayer.3） Bovine mammary epithelial cells showed positive reaction against cytokeratine 18 antigen.4） At passages 10 and 20 of cultivation,the cell chromosome number was 60 and the cells had normal diploid karyotype.In conclusion,tissue culture cells are able to obtain stable,functional bovine mammary epithelial cells,but are not immortalized cells.

关 键 词：奶牛乳腺上皮细胞 乳房炎 功能性 核型分析 

分 类 号：S823[农业科学—畜牧学]