The Study On Apoptosis And Its Signaling In Bovine Mammary Epithelial Cell Induced By Staphylococcus Aureus

The pathogenesis of persistent cow mastitis caused by Staphylococcus aureus was complex, and it was still not completely clarified. Pathogenic bacterium went through the milk ducts into the cow mammary gland contacting with bovine mammary epithelial cells first, so, their interaction had very important significance to the development of the mastitis process. Many pathogenic microorganisms could induce host cell apoptosis, and thus influenced the development of the disease process. Staphylococcus aureus could induce kinds of cells apoptosis including mononuclear cells and endothelial cells, which helped it to escape host immune mechanism and aggravated the disease infection. Whether the pathogenesis of persistent cow mastitis caused by Staphylococcus aureus was associated with its induction of apoptosis, and the apoptosis mechanism were unclear.We used the model of bovine mammary epithelial cells infected with three Staphylococcus aureus strains to observe the induction of bovine mammary epithelial cells apoptosis; to analyse the relevance of the Staphylococcus aureus virulence factors and the bovine mammary epithelial cells apoptosis; to explore the signaling mechanism of bovine mammary epithelial cells apoptosis induced by Staphylococcus aureus; to provide a theoretical basis for the understanding of cow mastitis mechanism, the design of antimicrobial drug and the development of vaccine.Firstly, bovine mammary epithelial cells were seperated from centrifuging fresh milk and digesting bovine mammary tissue by collagenase in vitro. Then, cells were characterized by RT-PCR, immunohistochemistry and karyotype analysis; Secondly, bovine mammary epithelial cells were infected with three Staphylococcus aureus strains, and then, we observed the morphological changes of the apoptosis cells through light microscope, electron microscope and nuclear staining, also, we detected the bovine mammary epithelial cells apoptosis rate through flow cytometry (Annexin V/PI); Thirdly, we identified the characteristics of Staphylococcus aureus virulence factors to find their relationship with apoptosis; Lastly, we detected the apoptosis related proteins caspase1/3/8/9by ELISA and Fas, FADD, TRAIL, Apo3L, LTa, IL-1β and TNF-a by Real-time PCR in bovine mammary epithelial cells before and after infection to explore the cell apoptosis signaling pathways.Results:1) We setted up the cultivation method of bovine mammary epithelial cells from centigrating fresh milk. Compared with collagenase digestion method, the milk method was more economical and simple, what’s more, the bovine mammary epithelial cells were pure without fibroblasts, which laid a solid foundation for further research.2) Staphylococcus aureus could induced bovine mammary epithelial cells apoptosis.3) Contacting the characteristics of Staphylococcus aureus with their apoptosis induction ability, we concluded that the hemolytic and infection ability of Staphylococcus aureus might be closely related to cell apoptosis.4) Analysis of caspase1/3/8/9、TRAIL、LTα、Apo3L and Fas showed that the apoptosis singnalling pathways of Bovine Mammary Epithelial Cells induced by Staphylococcus aureus might be Fas-ADD-caspase8-caspase3, while, mitochondrial cytochrome C-caspase9and caspase1-IL1β signaling pathways might not participate in the induction of apoptosis in this model.