Effect Of Methionine And Dipeptides Containing Methionine On Genes Expression Involved In Milk Protein Synthesis In Bovine Mammary Epithelial Cells

A model of bovine mammary epithelial cells (BMECs), which was cultured in vitro,was estiblished to study the effect of methionine and dipeptides containing methionine on genes expression involved in milk protein synthesis in BMECs and aminopeptidase content, to provide theoretical basis for investigating the mechanism of small peptides on milk protein synthesis and improving milk qulity.The study consisted of two parts:Experiment 1 was carried out to study the effects of different methionine concentrations and incubation time on the gene expression involved in milk protein synthesis in BMECs, and to select the optimal cultivation concentration and time.The BMECs was purified from mammary tissue of Holstein dairy cows and passaged when cell confluence reached to 80%. The study was designed as a single factor arrangement, and the third generation mammary epithelial cells were treated with 6 different concentrations of methionine (0,20,40,60,80 and 100μg/mL) at medium at 5% CO2,37 ℃ for 24 h,48 h or 72 h. Each treatment was run in sextuple. Results indicated that the vitality of BMECs and expression of αs1-casein (CSN1S1), κ-casein (CSN3) and β-lactoglobulin (LGB) were quadraticly increased with increasing methionine and the optimal expression was in 60 μg/mL methionine after 48 h incubation. However, the expression of β-casein (CSN2) was inhibited by the addition of free methionine. Cell proliferations of all treatments decreased after 72h incubation. It was concluded that the optimal methionine concentration and incubation time for BMECs vatability and gene expression related to milk protein synthesis were 60 μg/mL and 48h, respectively.Experiment 2 was conducted to study the effects of the equivalent free amino acids were instead with eight kinds of dipeptides containing methionine on the milk protein gene expression, small peptides transporterⅡ (PEPT2), aminopeptidase N gene (APN), aminopeptidase (APA) in the BMECs and the culture. The dipeptides (P-Met-Met,P-Met-Lys, P-Met-Trp, P-Met-Phe, P-Met-Thr, P-Met-Ile, P-Met-Leu P-Met-Val) equally insteaded of the corresponding free amino acid (F-Met-Met,F-Met-Lys, F-Met-Trp, F-Met-Phe, F-Met-Thr, F-Met-Ile, F-Met-Leu F-Met-Val). This experiment was consisted of three parts. The experiment included 3 parts. Part 1 was to study the effects of eight kinds of dipeptides on the milk protein gene expression, PEPT2,APN as well as APA inside and outside the BMECs. It was designed as sigal factor arrangement:control and 8 dipeptides groups.The results showed that the expression of CSN1,CSN2 and CSN3 was greater in P-Met-Met and P-Met-Lys than in other treatments, and a better expression was detected in P-Met-Met than P-Met-Lys group. The expression of CSN2 and CSN3 were down-regulated by P-Met-Thr, P-Met-Leu, P-Met-Ile and P-Met-Val.Part 2 was conducted to study the effects of the equivalent free amino acid with eight dipeptides on the gene expression of milk protein, PEPT2, APN, APA inBMECs or the culture. Cells were treated with 8 different free amino acid combination and control media. The result showed that the expression of CSN1S1 was significantly increased in P-Met-Met and P-Met-Lys groups versus other groups. Expression of CSN3 was lower in F-Met-Thr group than other treatments. Compared with control, F-Met-Ile significantly decreased the expression of PEPT2. But F-Met-Met significantly increased expression of PEPT2 than F-Met-Trp, F-Met-Leu, F-Met-Ile and F-Met-Val.The expression of APN was not effected by different trearments. The objective of part 3 was to study the effects of the dipeptides containing methionine equivalently instead of corresponding free amino acids on the milk protein gene expression, PEPT2, APN, APA in BMECs and-the culture. The results.detected that all treatments except P-Met-Val and P-Met-Leu upregulated the expression of milk protein gene and PEPT2 than the control. P-Met-Met had a best effect, and significantly increased expression of CSN1S1, CSN2 and CSN3 or tend to improve expression of PEPT2. P-Met-Trp, P-Met-Phe and P-Met-Lys also had a better effect in the expression of PEPT2.In conclusion, P-Met-Met, P-Met-Trp, P-Met-Phe and P-Met-Lys had better effect in the expression of milk protein gene, especially P-Met-Met best.