Translation Of Goat Mammary Epithelial Cell By 9,10-Dimethyl-1,2-Benzanthracene In Vitro

There are many reports on establishment of goat mammary epithelial cell line and description of its characters in Vitro. The experiment aims to establish a goat mammary cell line using two step carcinogenesis: 9,10-Dimethyl-1,2-benzanthracene(DMBA) as the carcinogen and 12-0-tet radecanoylphorbo-13-acetate(TPA) as the promoter. The methods of cell Feulgen dyeing and measuring DNA content of cell were also studied. The results as follows: 1. Primary culture of goat mammary gland cells could be derived by tissue culture and enzymes digest. Fibroblast and epithelial cells could be pured according to their different sensibility to trypsin. Complex cultures treated by 0.25% trypsin in Hanks` at 37℃firstly, the dispersed cells were mainly fibroblasts, The pured mammary epithelial cell could be obtained after 2~3 passages. 2. Using added 100 mL/L DMSO to D-MEM/F12 and 10%FCS as cryoprotectants, group the tissues to three: <0.3 mm, 0.3~0.5 mm and 0.5~1.0 mm.. the recovered time were 0,6 and 12 hours. The study showed using added 100 mL/L DMSO to D-MEM/F12 as cryoprotectants, keeping the size of tissue fragment less than 0.3 mm and 6 hours'equilibration before freezing had better effect on the freezing of goat mammary tissue. 3. The purified goat mammary epithelial cells kept normal characteristics until ninth passages. Majority of the cells were short shuttle-like or guboidal which looked like beebive; part of cells looked like big round flat cakes; part of the cells were long. Supernatant proteins of goat mammary epithelial cells were separated by SDS-PAGE and found there had three stripes in accordance with three standard protein stripes. The results proved that the cultured epithelial cells in Vitro could express goat casein. 4. Exposed the cells from tissue culture and enzyme digestion to D-MEM/F12 contained 5 ug/mL,10 ug/mL and 15 ug/mL DMBA for 36 h, 24 h and 12 h respectively, then cultured the cells in D-MEM/F12 contained 25 ng/mL TPA for two weeks. The result showed that the group contained 15 ug/mL DMBA was almost dead; the group contained 5 ug/mL5. DMBA showed the better effect of translation. The characters of translated cells were augmenting and flating, then the karyons of cell were concentrating, the size of cell were lessening at the same time; the array of cell were disorganized and the focus of translation appeared. 6. Using the method of Feulgen dyeing to measure DNA content of the translated cells, fixed in 95% ethanol for 15 minutes, hydrolyzed in hydrochloric acid for 10 minutes and dyed in Shiff for 20 minutes, the results were better. When dyed the cells on the plastic utensil directly, the last two step must omitted for influence the effect of dyeing.