In Vitro Culture And Application Of Bovine Mammary Epithelial Cells

Based on optimized culture of bovine mammary epithelial cells (BMEC), experiments wereconducted to investigate the effects on the growth of BMEC, the accumulation of lipid droplets incytosol, biosynthesis of fatty acid and gene expression of enzyme, with free fatty acid includingpalmitiate, steariate, oleiate and linoleiate in culture solution.Researching the effects of the in vitro culture for bovine mammary epithelial cells (BMEC) withdifferent method to separate, culture and purify. The results showed that the large numbers of BMECcould be separated from mammary tissue inoculation and digested with 0.25% trypsinase and 100u/mLhyaluronidase for 3h at 37℃.BMEC growed well in DMEM culture solution with 10% fetal calf serum,100ug/mL antibiotics, epithelial growth factor (EGF) (10ng/mL), insulin (0.5ug/mL), hydrocortisone(1ug/mL), transferring (5ug/mL), glutamine (0.3mg/mL) and pregnendione (1ug/mE). The lightmicroscope structure indicated that: there were different type cell in vitro, and most of them werepolygon, the ceils connected with each other properly, the boundary of the ceils was clear, but part oftheir boundary was not clear.Using the Western-blotting technique to detect the beta-casein protein secretion by BMEC in vitro,which can identify the normal physiological function of BMEC; Karyotype analysis identify the numberand figure of Chromosome of BMEC cultured in vitro. The results showed: containing beta-caseinprotein in culture medium of bovine mammary epithelial cells in vitro indicated that the mammaryepithelial cells with normal physiological condition; the number of chromosomes was normal, it was 60,and the karyotype agreed with the mammalian chromosome map, indicated that the cell line in vitroculture conditions had not transformatedWe examined the effects on the growth of BMEC, the accumulation of lipid droplets in cytosol andthe expression ofmRNA for ACC,FAS,FAT/CD36 and SCD, with free fatty acid including palmitiate,steariate, oleiate and linoleiate in a concentration-dependent manner from 100 to 400uM addition to themedium bathing BMEC. The results showed that palmitiate, steariate, oleiate and linoleiate inhibited thecell growth, which was positively correlated in the content as well as the saturation level of FA; Lipiddroplets formation were significantly increased by the addition of oleiate and linoleiate; ACC andSCDmRNA expression was inhibited by palmitiate, steariate, oleiate and linoleiate, but FAS,FAT/CD36mRNA expression was increased.