The Isolation And Identification Of Bovine Mammary Epithetical Cell And DBP-Induced Secrction Of β-Casein

Mammary gland bioreactors, which can produce a variety of pharmaceutical proteins utilizing the ability of animal breast tissues to synthesize and secrete milk, have become a very promising direction in biotechnology. Mammary epithelial cells are the specific cells with secreting function in mammary gland tissue. They retain a similar secreting function when isolated and cultured in vitro. Therefore, establishment of proper in vitro culture system for mammary epithelial cells has great significance for studying milk-secreting mechanism, and testing the function of mammary gland-specifically expressed vectors.In this study, bovine mammary epithelial cells were isolated, cultured identified, and treated with dimethyl phthalate dibutyl (DBP) to induce the expression of β-casein. The main contents of this study consist of the following two parts:1. Mammary epithelial cells were isolated from breast tissue in lactation cows, purified and identified. Three methods including explant culture, mechanical separation culture, and enzyme digestion culture were used to isolate primary cells. Differential attaching and detaching methods were combined to purify mammary epithelial cells. The results showed that the three methods all obtained mixture of fibroblast-like cells and epithelioid cells with good morphology. After purification, the fibroblast-like cells completely disappeared, and the remaining cells were all epithelioid cells showing a cobblestone shape. The purified cells were passed and subjected to morphology observation, growth curve plotting, and karyotype analysis. The results showed that the cells displayed epithelial cell morphology, had normal number of chromosomes, and grew vigorously. The expression of epithelial cell-specific markers was detected by immunofluorescence. The results showed that up to90%of the purified cell expressed epithelial cell-specific markers of keratin5,8and18. The expression of β-casein mRNA was detected by PCR. The results showed that the isolated cells could synthesize β-casein.2. The active monomer of Chinese herbal Vaccaria, β-Casein was employed to induce the expression of β-casein in the bovine mammary epithelial cells, with the medium containing insulin, hydrocortisone, insulin-like growth factor, and prolactin used as control. MTT analysis showed that0.4mg/ml,0.5mg/ml,0.6mg/ml,0.7mg/ml and0.8mg/ml DBP treatment promoted the proliferation of bovine mammary epithelial cells. Microscopic observation showed that DBP did not influence cell morphology. Flow cytometry measurement revealed that DBP treatment had no effect on cell cycle of the bovine mammary epithelial cells. Real-time PCR detection showed that0.5mg/ml DBP-induced cells had higher β-casein expression compared with either the non-treated cells or traditional control group.In conclusion, we successfully isolated and cultured bovine mammary epithelial cells, and established a new, more effective method to induce β-casein expression in bovine mammary epithelial cells. This study lays foundation for preparing transgenic mammary gland bioreactors and provides a powerful tool for testing the activity of mammary gland-specifically expressed vectors.