Effect Of Milk Fat Precursors On Subcellular Proteomics In Dairy Cow Mammary Epithelial Cells

The milk composition has directly affceted the quality of the milk. In this study, compaired with effects of milk fat precursors on abilities of dairy cow mammary gland epithelial cells (DCMECs) and the key enzyme gene expression of milk fat synthesis. Filter out the optimum concentration of milk fat precursors, which not only efficient to improve milk fat secretion abilities but also play an important role of regulatory key enzyme genes of milk fat synthesis, provide a reliable reference data of adding fatty acids in dietary. On this basis, through subcellular proteomics strategies and techniques research the effects of milk fat precursors on expression of DCMECs subcellular protein. Hope to be able to filter out the functional proteins which play a role of regulation in the process of milk fat synthesis and metabolism. Reference milk fat synthesis signaling pathway which have been known, analyze the link between the differentially expressed proteins and signaling pathways for the interpretation of the lactation mechanism to provide information on the subcellular level. Filter out the important function of the gene of bovine mammary development and lactation to improve milk quality, and promote dairy development has a great significance.In this experiment, DCMECs were cultured by the medthod set up previously in our laboratory. With CASY cytoanalyze detected effect of milk fat precursors on DCMECs’proliferation and viability. Through semi-automatic biochemical analyzers, high performance liquid chromatography (HPLC) and fluorescence quantitative PCR, the abilities of secretion of milk fat, lactose and beta-casein protein at different concentrations of sodium acetate or beta-hydroxybutyric acid sodium in DCMECs were detected respectively. Optimizing extraction method to the subcellular protein and conduct2-DE gel electrophoresis experiment, stained with silver nitrate to obtain high resolution, reproducible protein electrophoresis gel maps; combined with mass spectrometry (MALDI-TOF-MS) identity the effect of milk fat precursors on subcellular protein expression in DCMECs. By fluorescence quantitative PCR detection of these protein expressions in different lactating dairy cow mammary gland tissue, we found the expressions were positively correlated with the lactation ability of dairy cows.Experimental results show that:1. The milk fat synthesis precursors have different effectss on milk fat, lactose and β-casein protein which secretion from DCMECs, the expression of milk fat and lactose has improved significantly(P<0.05), but the expression of β-casein protein has decreased significantly; Through RT-PCR we detect differential expression of four key enzyme of milk fat synthesis in level of gene transcription; eventually filter out the best concentration of sodium acetate and β-hydroxybutyrate sodium on the role of DCMECs were4mM and0.75mM, respectively.2. Establishing and optimizing the experimental methods to the two-dimensional gel electrophoresis of DCMECs subcellular protein, get the protein spots and the good reproducibility of two-dimensional gel electrophoresis, that can provide an effective means for follow-up of DCMECs subcellular protein group.3. Through adding β-hydroxybutyrate sodium to DCMECs, analysis by two-dimensional gel electrophoresis combined with mass spectrometry, we get the successful of filtering and identifing four kinds of differences protein with great significance:Alpha-enolase(ENO1), Glucosidase (GANAB), Plastin3(PLS3) and Actin-related protein2/3complex subunit2(ARPC2), we identitied differential expression of four kinds of protein in level of gene transcription by RT-PCR; but after adding sodium acetate, we did not find significant differences in protein expression of DCMECs subcellular protein group, and its mechanism needs further study.4. We identitied differential expression of this four kinds of gene by RT-PCR, confirmed that these proteins were closely linked with the process of lactation in dairy cow mammary gland tissue.It is the first time to establish the method of study to subcellular protein group in DCMECs. Help explain milk fat synthesis and further to reveal the lactation mechanism of DCMECs. Prompted the mammary gland have the ideal balance mode of synthesis milk protein precursor, lactose precursor and milk fat precursor, it is worth further study.