奶牛乳腺上皮细胞的原代培养及其生物学特性分析

Primary Culture and Biological Characteristics Analysis of Bovine Mammary Epithelial Cells

旨在从奶牛乳腺组织中分离原代乳腺上皮细胞(bovine mammary epithelial cells,BMECs)并传代培养后探究其生物学特性。本研究从屠宰场采集健康泌乳奶牛乳腺并采用改进的酶消化法从乳腺中分离得到原代奶牛乳腺上皮细胞,通过形态学观察、免疫荧光以及染色体核型分析的方法对其进行鉴定。同时,研究第3、第6和第9代乳腺上皮细胞的生长曲线、群体倍增时间和冻存复苏活力,检测不同代次细胞分泌乳蛋白、乳脂、乳糖的功能及泌乳相关基因的表达。结果表明,所分离的奶牛乳腺上皮细胞纯度较好,细胞生长呈现S型,3个代次细胞的群体倍增时间依次为34.87、41.45和65.04 h,冻存复苏活力为88%~93%;在细胞分泌功能方面,诱导培养2 d后均能检测到酪蛋白、甘油三酯和乳糖,且各代次间无显著差异;此外,3个代次的细胞诱导后均能表达乳成分合成相关基因。本研究成功培养了原代奶牛乳腺上皮细胞,并证明直到第9代细胞仍然具有正常的生物学功能,为体外探究乳腺细胞增殖与分化机制提供了良好的试验材料和技术支撑。

This study aimed to isolate primary bovine mammary epithelial cells(BMECs)from mammary gland tissue and explore its biological characteristics after culture.In this study,the mammary glands of healthy lactating cows were collected from the slaughterhouse,the primary bovine mammary epithelial cells were isolated from the mammary glands by modified enzymatic digestion and verified by morphological observation,immunofluorescence and chromosome karyotype analysis.Meanwhile,the growth curve,population doubling time,resuscitation rate,secretion of milk protein,milk fat,lactose,and mRNA expression of lactation-related genes were investigated at the 3rd,6th,and 9th generations of mammary epithelial cells.The results showed that the isolated bovine mammary epithelial cells had good purity,and the cell growth presented an S-shaped curve,the population doubling time was 34.87,41.45 and 65.04 h of the 3 generations of cells respectively,and the resuscitation rate was 88%-93%.In terms of cell secretion function,the secretion of casein,triglycerides,and lactose were detected after induction culture for 2 days,and there were no significant differences among all groups.In addition,all the 3 generations of cells could express the lactation-related genes after induction.This study successfully obtained primary bovine mammary epithelial cells,and proved that the cells still had normal biological functions up to the 9th generation,which could provide experimental materials and technical support for exploring the mechanism of mammary cell proliferation and differentiation in vitro.