The Immune Study Of Peripheral Blood Mononuclear Cells In Cattle

Peripheral Blood Mononuclear Cells (PBMCs) contains two major classes of cells,monocytes andlymphocytes. Mononuclear phagocyte system (MPS) which is constituted by monocytes is an importantcomponent of the body's innate immune system, and MPS can present antigen which activateslymphocytes to cause acquired immunity. Acquired immunity are mediated by lymphocytes includeshumoral immunity and cell-mediated immune. In order to study the differentiation, immune functionand the inflammatory response to S.aureus mastitis of PBMCs. In this study,9healthy ChineseHolsteins were used to investigate the differentiation of monocytes, the function of lymphocytes and theimmune response to Staphylococcus aureus(S.aureus) of monocyte-derived macrophage and CD8Tlymphocytes in vitro. Studying the inflammatory response of PBMCs to actue phases of S.aureusmastitis using the real-time quantitative PCR. The result were as the follows:1Using magnetic bead separation technology, monocytes with the purity of90%isolated frombovine PBMC. Monocytes were cultured in vitro7d without cytokine stimulation and differentiated intomacrophage. By flow cytometry, cell surface antigen TLR2and CD172a of macrophages were increasedrelative to monocytes.2Transcription factor PU.1was cloned from PBMCs and constructing PU.1eukaryotic expressionvector. Recombinant plasmid was transfected H293T and bovine fetus fibroblasts, at24and48aftertransfection, bovine PU.1protein was detected by Western-blot successfully.3PBMCs were stimulated by PMA and PHA respectively, after48h there was a proliferation of CD8T cells,and bovine INF-gamma was detected using ELISA kit, there was an individual difference forinducing of INF-gamma.4Monocyte-derived macrophages were infected with heat-killed S.aureus, macrophages morphologyhas changed and surface antigen TLR2and CD172a were increased. With viable S.aureus infected,thereis no live S.aureus in macrophages.PBMCs were infected with heat-killed S.aureus,the intracellularINF-γ of CD8cells were significantly improved after PMA stimulation.5S. aureus-induced bovine mastitis model was built, The expression of IL-8, CD14, M-CSFR, TLR2,CCR2and CX3CR1in PBMCs at different time points for the acute phase of mastitis were measured byQ-PCR.The result showed that IL-8expression was significant increased on the first and second dayafter infection, the expression of CD14and CCR2were significant up-regulated on the first day,there was no significant changes in the expression of M-CSFR,TLR2and CX3CR1. We detected somaticcell count (SCC) and the levels of plasma LBP, The results showed that3days after infection weresignificantly increased, and reached the highest value on the first day.6The change of DNA methylation around STAT5-binding area in the far upstream promoter wasstudied during S.aureus mastitis in cows, the results suggested that DNA re-methylation of udder tissuecould be caused by S.aureus mastitis.