Primary Culture And Identification Of Bovine Mammary Epithelial Cell And Fibroblast Cell

To isolate and culture higher purity bovine mammary epithelial cells and fibroblast cells, the tissue block adherent method and milk separation method were used in this research. The cells obtained were purified by trypsin gradient digestion method, and specific molecules of this two kinds of cells that keratin8 gene, keratin 18 gene and (3-casein gene were identified by RT-PCR, and keratin7, keratin 18 and β-casein were identified by cell immunofluorescence staining.Then, analysis of karyotype and nutritional requirements, observation of Giemsa staining and determination of growth curve were performed on them. The experimental results showed that purity of mammary epithelial cells and fibroblast cells separated by tissue block adherent method respectively reached 96.3% and 99.7%, and purity of mammary epithelial cells got through milk separation method achieved 98.9%. Further identification demonstrated that mammary epithelial cells separated by both tissue block adherent method and milk separation method could transcript mRNA of keratin8, keratin 18 and (3-casein, and express keratin7, keratin 18 and β-casein. They had typical karyotype(2n=60), normal splitting proliferation characteristics. The viability of mammary epithelial cells separated by tissue block adherent method was higher than the one got through milk separation method. Culturing mammary epithelial cells need to add 20%FBS,200U two antibodies, hydrocortisone (1μg/ml) progesterone (1μg/ml), transferrin (5μg/ml), insulin (5μg/ml),200mmol L-glutamine, 10ng/ml EGF to DMEM/F12. Mammary fibroblast cells separated by tissue block adherent method could express vimentin, not P-casein, and had typical karyotype(2n=60), normal splitting proliferation characteristics, growing vigorously in basic medium, DMEM/F12 contained 10% FBS,so their viability was obviously higher than mammary epithelial cells. In conclusion, primary bovine mammary epithelial cells secreting P-casein protein and fibroblast cells that had typical morphology characteristics, higher purity, differentiation ability, normal splitting proliferation characteristics, were successfully separated and cultured, which will provide indispensable materials for subsequently establishing cell model of lactation and infection,et al.