Effect Of Interaction Between Amino Cids And STAT5A On Lactation Of Dairy Cow Mammary Epithelial Cells And Its Mechanism

China is a country of milk, however, milk quality and milk yield are very low, a high incidence of badly restricting the development of dairy industry of China, how to optimize the feed nutrition, especially the amino acids balance, improve the milk yield and quality as well as the content of lactoprotein have become an urgent problem. Ruminant animal amino acids requirement has been controversial topic world of nutrition. Application of ideal protein amino acids model in ruminant animal diets lag behind with respect to the pigs and poultry. Establish the optimal concentration ratio model of amino acids to guide the formulation of dairy cow’s diet has become a common pursuit of dairy researchers. Previous studies focused on the effect of different amino acids concentration ratios of diet on milk yield and content of lactoprotein, while taking DCMECs (dairy cow mammary epithelial cells, DCMECs) as the model, study on amino acids regulate lactation ability of DCMECs is less reported. This study takes lactating DCMECs as material, by the interaction method of nutrition and gene, study on the interaction between lactoprotein precursors amino acids and function gene STAT5A of lactoprotein synthesis, and the influence on lactation ability of DCMECs, which provide a theoretical basis for the optimization of dairy cows diet preparation.Primary dairy cow mammary epithelial cell was obtained through culture method of trypsin and collagenase I digestion, and the pured DCMECs own the characteristics of lactation detected by immunofluorescence, DCMECs within the10passages were as experimental model. The amino acids starvation time point of DCMECs was evaluated by qRT-PCR (Quantitative Real time PCR, qRT-PCR). At this starvation time point, content of lactoprotein by DCMECS synthesis was reduced almost to zero (p<0.01). Adding single essential amino acid to starved cells model, and determined Met (methionine, Met), Leu (leucine, Leu)、Phe (phenylalanine, Phe)、Thr (threonine, Thr)、Trp (tryptophan, Trp) could induce and open the re-synthesis of (3-Casein (beta casein, β-Casein), αsl-Casein (alphasl casein, asl-Casein), κ-Casein (kappa casein, κ-Casein), which the role of Met in promoting β-Casein maximum, followed by Thr, Leu and Phe (p<0.01). Using CASY-TT method for detecting cell viability and cell proliferation, found that Met and Leu promoted DCMECs viability and proliferation of the most significant (p<0.01). The date indicated that the five kinds of EAA could regulate the growth state of DCMECs to affect the synthesis of lactoprotein. At the same time, compared to the blank control group, these five kinds of amino acids could promote the expression of mTOR、S6K1、STAT5A、ELF5and Caveolin1measured by qRT-PCR (p<0.01), the date indicated that the five kinds of EAA could regulate the mTOR signaling pathway、STAT5A signaling pathway and the expression of Caveolin1to affect the synthesis of lactoprotein; the mRNA expression of the function gene SREBP1and GLUT1related to synthesis of milk fat and lactose, the key gene Cyclin D1related to cell proliferation were all up-regulated (p<0.01), the date indicated that the five amino acids could regulate the expression of SREBPland GLUT1to affect the synthesis of milk fat and lactose, to regulate Cyclin D1to affect the cell proliferation. The contents of triglyceride and lactose were also increased (p<0.01) compared to the blank control group, the data indicated that the five amino acids could promote the synthesis of milk fat and lactose of DCMECs. In order to obtain the optimal concentration ratio model of amino acids for lacoprotein synthesis, these five kinds of amino acids were five factors and three levels of orthogonal test was launch. The optimal concentration ratio model of amino acids:Met:Leu:Phe:Thr:Trp=158:95:1341:427:228μmol/L. Under this model, cell viability and cell proliferation were increased significant detected by CASY-TT (p<0.01), which indicated that the optimal concentration ratio model of amino acids could regulate cell viability and proliferation to affect lactation of DCMECs. Through qRT-PCR to detect the influence on the expression of function genes related to lactation by the optimal concentration ratio. qRT-PCR results showed that compared to the blank control group, except tryptophan aminoacyl tRNA synthetase decreased, expression of LeuRS, RARS and other essential amino acids in the aminoacyl tRNA synthetase were raised (p<0.05or <0.01), mTOR、S6K1、 PRLR、JAK2、STAT5A、ELF5、Caveolin D1、SREBP1(p>0.05). AKT1、GLUT1and Cyclin D1all up-regulared (p<0.05or p<0.01), which indicated that the optimal concentration ratio model of amino acids could regulate the mRNA expression of above genes to affect lactation in DCMECs. Through Western blotting to detect the influence on the expression of function genes related to lactation by the optimal concentration ratio. Western blotting results showed that compared to the blank control group, mTOR、p-mTOR、S6K1、p-S6K1、STAT5A, as well as SREBP1、AKT1、GLUT1and Cyclin D1, expression of these proteins related to lactation were all up-regulated (p<0.05or P<0.01), which indicated that the optimal concentration ratio could regulate aminoacyl tRNA synthetase、mTOR signaling pathway and STAT5A signaling pathway to affect the synthesis of lactoprotein, regulate the mRNA expression of SPEBP1、AKT1、GLUT1to affect the synthesis of milk fat and lactose, regulate the mRNA expression of Cyclin D1to affect DCMECs proliferation. Testting the triglycerides and lactose secreted into the culture supernatant, the results showed that, compared to the control group, the secretion of triglycerides and lactose (p <0.01) increased significantly.Analyses on the differentially expressed key genes related to lactation in mammary gland tissues and combined with differences in gene expression profiling of dairy cow mammary tissues, screening out the lactoprotein synthesis key gene-STAT5A as the object of study, to study the regulation mechanism of interaction between different concentration ratios of amino acids and STAT5A gene. Using gene silencing technology, qRT-PCR to detect STAT5A gene silencing efficiency, after transfection24h and compared to the negative control group, STAT5A mRNA expression in inhibition group was reduced by80%(p<0.01). The effect of different concentration ratios of amino acids and STAT5A gene silence on the expression of key genes in lactation signaling pathway was detected by qRT-PCR. Compared to the negative control group after transfection24h, the expressions of STAT5A、SREBP1、GLUT1、Cyclin D1and β-Casein were all down-regulated in the STAT5A inhibition group (p<0.05); compared to the STAT5A inhibition group, the expression of SREBP1、Cyclin D1and β-Casein were up-regulated in the AA2+inhibition group (p<0.05), but the expression of STAT5A、SREBP1、GLUT1、Cyclin D1and β-Casein were down-regulated in the AA3+inhibition group (p<0.05). The effect of different concentration ratios of amino acids and STAT5A gene silence on the expression of key genes in lactation signaling pathway was detected by Western blotting. Compared to the negative control group after transfection24h, the expression of p-STAT5A、STAT5A、SREBP1、GLUT1、Cyclin D1and β-Casein were all down-regulated (p<0.05), compared to the STAT5A inhibition group, the expression of SREBP1and Cyclin D1were up-regulated (p<0.05), but for the expression of p-STAT5A, STAT5A and of β-Casein less affected, the expression of SREBP1was up-regulated in the three amino acids adding group, p-STAT5A、STAT5A, GLUT1and Cyclin D1were down-regulated significantly in AA3+inhibition group. Testting the triglycerides and lactose secreted into the culture supernatant, the results showed that, after transfection48h compared to the control group, the secretion of triglycerides and lactose decreased significantly in the STAT5A inhibition group (p<0.05), compared to the STAT5A inhibition group, DCMECs stimulate the secretion of triglyceride and lactose in the AA2+inhibition group (p<0.05), however, the secretion of triglyceride and lactose had little change in both AA1+inhibition and AA3+inhibition groups (p>0.05). In this research, eukaryotic expression vector pEGFP-C1-Stat5a was constructed; the transfection efficiency was detected by qRT-PCR after transfection24h, compared to the negative control group, STAT5A mRNA expression in pEGFP-C1-Stat5a group was increased15.5times, the difference was significant (p<0.01). The effect of different concentration ratios of amino acids and STAT5A overexpression on the expression of key genes in lactation signaling pathway was detected by qRT-PCR. Compared to the negative control group after transfection48h, the expression of SREBP1, GLUT1, Cyclin D1and β-Casein were increased at different degree in the pEGFP-C1-Stot5a group (p<0.05); Compared to the pEGFP-C1-Stat5a group, the expression of SREBP1、GLUT1and Cyclin D1were up-regulated in the three amino acids adding groups, however, the expression of STAT5A、Cyclin D1and β-Casein decreased significantly in the AA3+pEGFP-C1-Stat5a group (p<0.05), SREBP1and GLUT1had little change (p>0.05). The effect of different concentration ratios of amino acids and STAT5A overexpression on the expression of key genes in lactation signaling pathway was detected by Western blotting, compared to the negative control group after transfection72h, the expression of SREBP1, GLUT1, Cyclin D1and β-Casein were up-regulated in the pEGF-P-Cl-Stat5a group; compared to the pEGFP-CI-Stat5a group, the expression of SREBP1、GLUT1and Cyclin D1were up-regulated in the three amino acids adding groups (p<0.05), however, at this amino acid concentration ratio model not being able to continue to enhance the expression of STAT5A protein (p<0.05), the synthesis of β-Casein also be suppressed (p<0.05). Detection of triglycerides and lactose secreted into the culture supernatant, after transfection72h compared to the control group, the results showed that the secretion of triglycerides and lactose increased significantly in the pEGFP-Cl-Stat5a group (P<0.05), compared to the pEGFP-Cl-Stat5a group, triglyceride and lactose were also significantly increased with the increase of the content of amino acid concentration in the three amino acids adding groups (p<0.05), but there was no significantly difference among the three groups (p>0.05).The results of this research will provide valuable data and theory foundation for the study content of molecular nutrition of lactating dairy cow.