| There are few reports on establishment of goat mammary epithelial cells until now. The present experiment aims to establish a goat mammary cell line for examining mammary special expression vector and other biological research. The culture methods and characteristics of goat mammary epithelial cell were also studied. The results are as follows: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癈, the majority cells harvested were mammary epithelial cells. The pured mammary epithelial cell line could be obtained by 2~3 passages.2. Collegenase I and Hyaluronidase were appropriate enzymes to dissociate goat mammary gland tissue. 1mm3 goat mammary gland tissue fragments incubated in Collegenase I and Hyaluronidase solution (150IU/ml and lOOIU/ml in 10%FCS RPMI1640, especially) 4h at 37℃, could produce enough dispersed cells to explant.3. Primary culture of goat mammary gland cells could be derived by tissue of its mammary gland in culture dishs treated with rat tail collagen in 5~10d, while no mammary epithelial cells grew in plastic petri dishs without collagen treatment in the same duration. The rat tail collagen has no effect on the growth of the passaged cells or dispersed cells digested with enzyme.4. Using RPMI1640 containing 10%FCS, lOOIU/ml Penicillin and lOOIU/ml Streptomycin as basic media, several different growth factors such as insulin, hydrocortisone, insulin-like growth factor, EOF and so on, were added and selected to culture goat mammary epithelial cells. The result suggested that RPMI1640 supplemented with 10%FCS, 5ug/ml insulin, 5ng/ml hydrocortisone, 100ng/ml insulin-like growth factor, lOng/ml EGF and 5ug/ml ITS had the best protective effect to the growth of epithelial cells.5. Using RPMI1640 and DMEM/F12 as basic media, the growth curves of goat mammary epithelialcells in these media supplemented with growth factors were made. The goat mammary cells were in lag phase in first two days, then they entered log phase and persisted 5-6 days; 8 days later, they stayed at plateau phase for about 4 days. The DMEM/Fu medium added with hydrocortisone, IGF-1, EOF and ITS is fit to epithelial cells.6. The goat mammary epithelial cells were frozen in DEME/F12 with serum and antibiotics by adding with cryogen of 10% DMSO and 15% Glycerol, respectively. Attachment rates of the thawed cells after 48h were 71.95% and 82.21%, respectively. The result showed 10% DMSO and 15% Glycerol had the same protective effect. When serum content of cryogen reache 10%, 30% and 50%, their attachment rates were 72.53%, 82.21% and 90.37%, respectively. This demonstrated that the treatment with 50% PCS and 10% DMSO had the best protective effect, which can meet the needs of passaging and long-term preservation of these cells.7. The goat mammary epithelial cells keep normal characteristics by 7th passages with the medium of DMEM/Fi2 and there are no sign of apoptosis. Perspective electron microscope showed the difference between the 6th and the 7th passage is few. The nuclear structure is clear while the surface of some cells nuclear has bulges and hollows. The nucleolus are rather big. There are rough endoplasmic recticulums and mitochondria in the cytoplasm. The presence of lipid droplets and Golgi vesicles attest to the vigorous secretory function of mammary epithelial cells. Many vacuoles move toward cell membrane and some of them contact and fuse with membrane from a pore so to let out the cordents. These results suggest goat mammary epithelial cells proliferate vigorously at the 7th passage in vitro.8. Using the rabbit anti-goat-casein-serum as the standard antibody, the casein of mammary epith... |
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