| The quality and safety of milk has become a key problem to sustainable development of the dairy industry in our country. Milk protein is a main component in milk, which is not only a sign of dairy industry core competitiveness but also related to dairy quality and safety. The content of protein in the milk is generally lower in our country at present (average 2.8%). It not only cannot reach the genetic potential of cows but also even lower than the national standard (2.95%). Therefore, it has become a major demand for sustainable and healthy development of dairy industry to based on reality of feed resources in China, expounds the mechanism of milk protein synthesis and regulation, establish the theory and methods of improving the quality of milkThe effects of insulin, prolactin and hydrocortisone on the synthesis of milk protein in bovine mammary epithelial cells were studied in vitro, and the enzyme digestion was used to cultivate the cells.Part I:Healthy Holstein cows from Yangzhou university experimental farm with mastitis-free were choosed to take breast tissue sterile. The enzyme digestion was used to separating, purificating and culturing the bovine mammary epithelial cells. The results showed that the cells were well-grown in the DMEM/F12 culture medium with 10% fetal bovine serum, 300 IU/mL penicillin,300 IU/mL streptomycin,25 ug/mL amphotericin B, epidermal growth factor, ITS, hydrocortisone, prolactin and so on. The microstructure of cells showed that most of the cells morphological characteristics were oval, round and polygonal within eight generations. The cell was connected piece each other but has visible boundaries. The results of fluorescent immune cell staining analysis showed that almost all the cells were positive cells, which confirming that the cells with typical morphological characteristics were received really came from the bovine mammary epithelial cells with the normal physiological function. It also showed that the method of enzyme digestion was an effective method to obtain the pure bovine mammary epithelial cells.Part II:The experiment was based on the previous obtained cell lines. The single factor experimental design for 5 levels were used respectively to study the effects of different concentration of prolactin (0,5,50,500,5000 ng/mL), insulin (0,5,50,500,5000 ng/mL) and cortisol (0,1,10,100,1000 ng/mL) on bovive mammary epithelial cells growth (CCK 8 cell proliferation and Annexin V-FITC inmanufacturing apoptosis detection kit), cell signaling pathways and hormone receptor genes expression related to bovine milk protein synthesis (real-time PCR) and the influence of the relative contents of casein. The results showed that, firstly, the cells proliferation, s-PRLR gene expression, kappa-casein and total casein production were promoted significantly by prolactin (P<0.05). With the increase of prolactin dose, the receptor and CSN3 genes expression, the relative content of kappa-casein and total casein were increased before 50ng/mL. After that, the expression and the relative content have decreased instead. Secondly, the cells proliferation was promoted by 5-500 ng/mL insulin significantly. The relative content of kappa-casein and total casein was improved by 5-5000 ng/mL insulin, and CSN3 gene expression was significantly improved 50 ng/mL insulin. To INSR gene xpression, with the increase of insulin before 50ng/mL, the gene expression was increased, after that, the expression of INSR was decreased instead. Finally, the cells proliferation was not effect by HYD before 24h, but it was inhibited significantly by 1000 ng/mL hydrocortisone after 24h. The CSN3 gene expression was increased by 1-100 ng/mL HYD significantly, and NR3C1 gene expression was increased significantly 1 ng/mL and the relative content of kappa-casein and total casein was increased by 1-10 ng/mL HYD significantly.Part III:The interaction of insulin, PRL and HYD were studied in the experiment. The effect of interaction of hormones on bovine mammary epithelial cell proliferation and apoptosis, on hormone receptor genes and the genes expression in mTOR and JAK-STAT signal pathway, on the relative contents of kappa-casein and total casein. The L93 (4) orthogonal experiment design and cryopreserved cow mammary epithelial cells were used in this study. The results showed that, firstly, the cells proliferation was increased significantly by the synergy of the three kinds of hormone. Secondly, the mTOR, Rheb, PKB, PRAS40, STAT5a and P53 genes expression was increased by 5 ng/mL prolactin,50 ng/mL insulin and 1 ng/mL HYD significantly, but the different influence degree was there on different genes expression of mTOR signal pathway. But to JAK-STAT signal pathway, the priorities is C>B>A.Thirdly, to s-PRLR gene expression, the optimal combination of the three kinds of hormones is A1B2C2, and the priorities to the result is OB>A.To NR3C1 gene expression, the optimal combination is A2B1C3, the priorities to the result is A>B>C. To INSR gene expression, the optimal combination is A2B2C1, the priorities to the result is A>B>C. Finally, the optimal combination of the three kinds of hormone to increase the CSN3 gene expression, the relative content kappa-casein and total casein is A1B2C2.Part IV:Single factor 4 levels experimental design was used in this study. Namely, the control (A1B2C2), LY294002 group (A1B2C2+15ng/mL LY294002), AG-490 group (A1B2C2+15ng/mL AG-490) and double blockers (A1B2C2+15ng/mL LY294002+15ng/mL AG-490). The order of this experiment is to verify the previous conclusion by determining the hormone receptor gene expression, the positive regulatory genes expression related to the two signal pathways, the casein genes expression and the relative content of casein in bovine mammary epithelial cells. The results showed that the cells proliferation was inhibited and cell apoptosis was induced by inhibitors. It shows that the physiological basis for milk protein secretion was based mainly by promoting cells proliferation and inhibiting cells apoptosis by prolactin.Inhibitors inhibited the positive regulatory gene expression related to mTOR and JAK-STAT signaling pathway significantly, which showed that hormones mainly by promoting the positive regulatory gene expression related to mTOR and JAK-STAT signaling pathway related to regulation and promotion the casein gene expression, and then promoting the synthesis of milk casein in bovine mammary epithelial cells, and then improving the content of casein in milk.In terms of the two signaling pathways, JAK-STAT signaling pathway is more critical, that means the casein synthesis in bovine mammary epithelial cells was blocked obviously once the JAK-STAT signaling pathway was blocked.Therefore, it was concluded that either alone or synergy of insulin, hydrocortisone and prolactin could promote the growth and milk protein synthesis in the bovine mammary epithelial cells. The optimal dose of insulin, hydrocortisone and prolactin to milk protein synthesis in bovine mammary epithelial cells was 50ng/mL,50ng/mL and lng/mL. The optimal synergy combination to milk protein synthesis in bovine mammary epithelial cells was 50 ng/mL insulin, 5ng/mL prolactin and 1 ng/mL hydrocortisone. The hormone-hormone receptor-JAK/STAT signaling pathway and the hormone-hormone receptor-mTOR signaling pathway were the key action mechanism of hormones to regulate the the growth of cells and synthesis of milk protein in bovine mammary gland. |
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