围产蒙古牛中性粒细胞类固醇激素受体对Toll样受体信号通路膜分子表达的作用研究

Mongolian cattle is a unique breed in northern region of China, and it possesses the outstanding ability of resilience and disease resistance, especially, the incidence of perinatal disease was significantly lower than cows. At present, the significant correlation has been widely confirmed between perinatal immune suppression between high incidence of perinatal diseases, and steroid hormone is an important inducing factor and neutrophils (PMN) dysfunction is a key factor. However, no research was performed on perinatal immune suppression in Mongolian cattle till now, especially, the steroid hormone receptor expression and mediation of them on membrane signaling molecules expression in PMN. The project firstly intends to determine the pattern of estrogen (E), progesterone (P) and glucocorticoid (GC) in peripheral blood of perinatal Mongolian cattle, and then analyze the hormone nuclear and/or membrane receptor mRNA and protein expression and cellular localization of them, and expression of TLR2/4 signaling pathway-associated membrane molecules mRNA and protein. Basis on the above results, the mediated effects of the genomic pattern and non-genomic pattern of hormone receptor on PMN activity was evaluated in vivo and in vitro. Ultimately, it is clarified that the signal contact of steroid hormones, hormone nuclear and/or membrane receptor and TLR2/4 signaling pathways, and the role which plays on signaling networks of PMN activity. The expected result will fill the blank of immune physiology research fields in Mongolian cattle, and provides the scientific reference for revealing the characteristics of disease resistance in the Mongolian cattle.

蒙古牛是我国北方特有的牛品种，其抗逆抗病性突出，围产疾病发病率显著低于奶牛。奶牛围产期免疫抑制与疾病高发密切相关已被广泛证实，其中类固醇激素是重要诱因，中性粒细胞（PMN）机能障碍是关键因素。目前有关蒙古牛围产期免疫抑制，特别是PMN类固醇激素受体表达及其对膜信号分子介导效应的研究尚属空白。本项目拟在确立围产蒙古牛外周血雌激素（E）、孕酮（P）和糖皮质激素（GC）"范型"基础上，通过分析PMN在体内/外条件下的激素核/膜受体mRNA与蛋白表达及亚细胞定位、TLR2/4及其通路膜分子mRNA与蛋白表达，以评价激素受体"基因组模式"和"非基因组模式"对PMN活性的介导效应，并阐明围产蒙古牛外周血类固醇激素、激素核/膜受体与TLR2/4信号通路膜分子的信号联系及其与PMN活性的关系。预期成果将填补蒙古牛免疫生理研究领域的空白，并可能为揭示蒙古牛抗病特性提供科学的理论参考。

基于外周PMN类固醇激素受体表达及其介导免疫抑制的研究目前尚属空白，本项目以蒙古牛为研究对象，取得如下成果。①围产期蒙古牛外周血GC、E2和P4变化规律，与围产期奶牛GC、P4、E2的经典动态变化规律相近。②外周血白细胞指标变化：分娩前白细胞总数、PMN总数和嗜酸性粒细胞总数均显著增加，约在分娩前7-10天升至最高值，后降至分娩时最低，分娩后逐渐升高至分娩前7天水平；淋巴细胞总数变化不明显。③首次在基因转录和蛋白表达水平上同时确定PMN、淋巴细胞的mPRs（mPRα、mPRβ、PGMC1）表达，对于结束牛PMN是否mPRs表达的学术争议提供了有力的科学依据。④验证了P4、GC特异性核受体的交互介导作用，发现了阻碍P4核受体途径显著改变mPRs、GR s基因转录水平，阻碍GC核受体途径也显著改变nPRs、mPRs的表达。mPRs介导GC的细胞效应具有显著的浓度和时间依赖性特征。⑤发现了低浓度P4、GC快速显著上调TLR2、TLR4基因转录的非基因组调节效应、高浓度P4、GC显著抑制TLR2、上调TLR4基因转录的信号转导。. 总之，围产牛、体内注射、体外诱导的结果表明，GC、P4能够通过膜受体介导的快速基因组途径、核受体介导的基因组途径调节细胞活性，调节效应具有GC、P4浓度和时间依赖性特征，而体内注射、体外诱导的分析结果与围产牛并非完全一致。这提示我们，仅仅以围产状态下免疫细胞的GC、P4特异性核/膜受体的表达水平、TLR2/4以及NFκB等炎性信号表达及磷酸化水平评价GC、P4在围产期免疫细胞活性抑制中的诱导作用，不能完全阐明围产期GC、P4、E2浓度持续交互动态变化这一复杂背景下其特异的细胞活性调控效应。本研究结论的意义在于确定了牛PMN和淋巴细胞的mPRα、mPRβ和PGMC1表达，系统阐释了性类固醇激素经由基因组途径、基因组途径调节细胞活性浓度和时间依赖性特征，为阐明围产牛外周性类固醇激素调节免疫细胞活性的信号机制提供了新的探究点