The Culture In Vitro Of Mammary Epithelial Cell In Goat

Culture system in goat mammary epithelial cell was established to study the molecular mechanism of proliferation and differentiation in goat mammary epithelial cell. The mammary tissues from healthy Xinong saanen goat during lactation have been cultured in vitro.The basic medium is D-MEM/F-12 and 10% fetal calf serum,and EGF(0.01mg/L),hydrocortisone (1mg/L),P(1mg/L), ITS(5mg/L),penicillin(0.05g/L) and streptomycin(0.05g/L) were added. The cell biological propertied were observed and investigated by observing cell population doubling time and cell growth curve. Goat mammary epithelial cell lines were obtained by culturing tissue fragment. The appearance of these cells was studied by light microscope. 1.Primary culture of goat mammary gland cells could be derived by outgrowth of migrating cells from fragment of tissue. Fibroblast and epithelial cells could be pured according to their different sensibility to trypsin. Complex cultures treated by 0.25% trypsin in Hanks` for 5~7min at 37℃, the dispersed cells were mainly fibroblasts,then the rest treated by 0.15% trypsin-0.02% EDTA in Hanks`for 5~8min at 37℃, the majority cells harvested were mammary epithelial cells. The pured mammary epithelial cell line could be obtained by 2~3 passages. 2.Using RPMI1640 containing 10%FCS, 100IU/ml Penicillin and 100IU/ml Streptomycin as basic media, several different growth factors such as insulin, hydrocortisone, insulin-like growth factor, EGF and so on, were added and selected to culture goat mammary epithelial cells. The result suggested that RPMI1640 supplemented with 10%FCS, 5μg/ml insulin, 5μg/ml hydrocortisone, 100ng/ml insulin-like growth factor, 10ng/ml EGF and 5μg/ml ITS had the best protective effect to the growth of epithelial cells,and doubling time was 34 hours. 3.The growth curve indicates that, after inoculation, the BMEC resort 2 days, stay in stagnate phase for 4 days, them came into logarithmic growth phase. 4.The suitable frozen medium for BMEC is DMEM/F12 added 10% DMSO and 30% FCS. The temperature should be dropped from 4℃to -25℃at the rate of -1℃/min,30 minutes later, put the cells into liquefied nitrogen for long term preservation. 5.The fibroblast cell and the goat mammary epithelial cells grow in different district. The goat mammary epithelial cells were cultured in vitro through single-cell afloat, the cells formed island monolayer aggregates, the cells propagated and formed dome-like construction. Nucleolus is circular or oval,two to four nucleoluses.