Dysfunction of neutrophil during parturition and proteome analysis of placenta and serum in cow

本项目的目的是确定和表征奶牛分娩后中性粒细胞功能降低的内部因素，因为奶牛乳房炎有时会在分娩期间发生。在实验中，对牛中性粒细胞激活的信号转导途径进行了分析。作为牛中性粒细胞NADPH氧化酶激活的重要分子，cPKC、p38MAPK、ERK、PI3K和nPKC(pKCδ)在奶牛分娩过程中不发生变化。此外，本研究还利用双向电泳法等蛋白质组学技术，试图找出奶牛分娩后免疫功能下降的原因。蛋白质组分析表明，血清和胎盘中的乳铁蛋白、转铁蛋白、牛妊娠相关糖蛋白(BPAG)和酮体是牛中性粒细胞NADPH氧化酶活性的抑制因子。此外，为了阐明乳铁蛋白抑制中性粒细胞功能的机制，我们还观察了乳铁蛋白对内毒素或刀豆蛋白A诱导的小鼠巨噬细胞和猫单核细胞中干扰素-γ、白介素1β、肿瘤坏死因子-α、白介素4O和诱导型一氧化氮合酶基因表达的影响。结果表明，乳铁蛋白可降低脂多糖或刀豆蛋白A诱导的干扰素-γ和IL-1β的mRNA表达，但不影响肿瘤坏死因子-α、IL-12p4O和iNOS的表达。在HL60细胞中加入乳铁蛋白可减少骨髓细胞向中性粒细胞的分化。这些结果表明乳铁蛋白与炎症和免疫系统的乳腺调节有关。

The purpose of this project is to identify and characterize internal factors to reduce neutrophil functions after parturion in cows, since bovine mastitis sometimes occurs during parturition. In the experiments, the analysis of signal transduction pathways for activation of bovine neutrophils was performed. As important molecules for activation of NADPH oxidase in bovine neutrophils, cPKC, p38MAPK, ERK, PI3K and nPKC(pkcδ) were identified, but these factors did not changes during parturition in the cow. Furthermore, I tried to identify causative factors to reduce immune functions of cow after parturition by using two dimensional electrophoresis and other proteome technique. The proteome analysis showed that lactoferrin, transferrin, bovine pregnancy related glycoprotein (bPAG) and keton body in serum and placenta playd as suppressive factors for NADPH oxidase activation of bovine neutrophils. Moreover, to clarify the mechanism of this suppressive effects on neutrophil function, the effects of lactoferrin on LPS-or conA-induced expression of mRNA of INF-gamma, IL-1beta, TNF-alpha, IL12p4O and iNOS in mouse macrophage and feline monocyte was examined. The results indicated that the treatment of lactoferrin decreased LPS-or conA-induced expression of mRNA of INF-gamma and IL-1beta but did not affect that of TNF-alpha, IL12p4O and iNOS. The addition of lactoferrin to culture medium was showed to reduce differentiation form myeloblast to neutrophil in HL60 cells. These results suggest that lactoferrin was associated with regulation of inflammation and immune system mammary gland.