Effect Of Free Amino Acids And Peptides On The Expression Of Casein α_s1 Gene In Cultured Bovine Mammary Epithelial Cells

This study was conducted to establish an in vitro model for culture of lactating bovine mammary epithelial cells. Comparison was firstly made of isolation methods for collection of bovine mammary epithelial cells, and then the optimal method was chosen to study the effects of bipeptides, free amino acids and the proportion of lysine and methionine on the expression of caseinα_s1 gene of cultured mammary epithelial cells.1. Comparative research of methods for collection of bovinemammary epithelial cells.The growth and activity of mammary epithelia cells that were obtained by digestion, mammary tissue explant culture or separated directly were measured by MTT cell proliferation assay for a culture period of seven days.Cells obtained by digestion with collagenase type I or type III had low activity and could not grow or proliferate normaly when they were cultured. Large number of cells could not be collected through the method separated directly, but cell mass could be obtained. These cell mass suspended in medium but did not attached entirely to the bottom of culture palte, so they appeared low activity and could not grow or proliferate well, too. Mammary tissue explant culture was the optimal way to obtain the mammary epithelial cells. The cells obtained by this way presented a monolayer cobblestone epithelial like morphology in MTT cell proliferation assay, and the OD level of cells was significantly higher than that obtained by the other two methods (P<0.01). The curve of OD level presented an S shape, which followed the rule of cell growth.2. Effects of methionine and bipeptide on expression of caseinα_s1 oncultured bovine mammary epithelial cellsThis experiment was carried out to investigate the effects of methionine and bipeptide on cultured bovine mammary epithelial cells. The methionine-modified medium consisted of 0-100μg/ml of methionine. The expression of caseinα_s1 gene was increased with adding level of methionine from 0 to 60μg/ml, and then decreased. The estimated optimal adding level of methionine was at 57μg/ml. Expression of caseinα_s1 gene was much higher when 60μg/ml methionylmethionine or 120μg/ml methionyllysine was added instead of corresponding free amino acids (P<0.01). SDS-PAGE showed that the concentration of casein in culture medium were higher when methionylmethionine or methionyllysine were added instead of corresponding free amino acids. These results indicated that mammary epithelial cells can utilize methionylmethionine and methionyllysine for milk proteinsynthesis with a higher efficiency than methionine or methionine and lysine.In order to investigate the optimal concentration of methionylmethionine up-regulating the expression of caseinα_s1, 60, 70, 80, 90,100 and 110μg/ml of methionylmethionine was added to the medium. The expression of caseinα_s1 gene was increased with no significantly difference when adding level of methionylmethionine change from 60 to 70μg/ml, and then decreased significantly with the increasing methionylmethionine concentration from 80-110μg/ml. 3. Effect of proportion of lysine and methionine on expression ofcaseinα_s1 on cultured bovine mammary epithelial cellsLysine and methionine (57μg/ml ) were added to the culture medium with proportion of 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 and 4.5:1 respectively. The cells attached to the bottom of culture plate were digested with trypsin and EDTA after culture for 48 hours. Expression of caseinα_s1 gene were detected through the RT-PCR method. Expression of caseinα_s1 gene was increased with the increasing proportions of lysine and methionine from 1:1 to 3.5:1, and then decreased. Expression of caseinα_s1 gene with proportion of lysine and methionine at 3:1 showed significant difference with those at the proportions lower than 3:1, but there were no significant difference with those at the proportions higher than 3:1.In summary, mammary epithelial cells collected by mammary explant culture can grow and proliferate normaly. Expression of caseinα_s1 gene was up-regulated when methionylmethionine or methionyllysine were added instead of corresponding free amino acids. Different proporations of lysine and methionine in medium can affect the expression of casein gene with the optimal proporation at 3.5:1.