microRNAs 在奶牛乳腺上皮细胞抗金黄色葡萄球菌免疫反应中的调控机理

Staphylococcus aureus (S. aureus) was one of the most frequently isolated etiological agents from cows with mastitis. MicroRNAs (miRNAs) have regulatory roles in bovine immunity of mammary gland. To date, however, the regulatory roles and molecular mechanisms of miRNAs in immune responses by bovine mammary epithelial cells (bMECs) to S. aureus isolated from bovine mastitis have remained poorly understood. Our previous findings demonstrated that nine different virulence gene patterns were found in a total of 35 S. aureus isolates obtained from mastitis milk samples in Ningxia province of China. In addition, there was great variation in RAPD genotypes in S. aureus isolates not only among different farms, but also within the same herd in Ningxia province. Furthermore, an obvious relationship was existed among the RAPD genotypes, SAg toxin genes and severity of bovine mastitis. The objective of this project is to investigate regulatory roles and molecular mechanisms of miRNAs in immune responses by bMECs to S. aureus isolated from bovine mastitis and provide a theoretical basis for elucidating the pathogenesis of bovine mastitis caused by S. aureus. MiRNAs expressions in bMECs challenged with S. aureus will be characterized by next generation sequencing at different time points. Using the overexpression or silencing of genes, miRNAs associated with immune response by bMECs will be screened. The target gene will be screened by means of luciferase reporter assay and the signaling pathway mediated by miRNAs to regulate immune response by bMECs to S. aureus will be identified. Using immunofluorescence analyses, qPCR, Western blot and ELISA, the regulatory roles of the signaling pathway mediated by miRNAs in host defense to S. aureus will be verified in animal and cell models.

金黄色葡萄球菌是奶牛乳腺炎的主要病原菌。miRNAs 调控奶牛乳腺免疫，但它在奶牛乳腺上皮细胞抗金葡菌免疫反应的调控作用和分子机制不清楚。我们前期研究结果表明，宁夏地区奶牛金葡菌分离株具有9种不同毒力基因组合；RAPD基因分型存在明显差异；超抗原毒素基因、RAPD基因分型和菌株致病性之间呈高度正相关。本项目拟采用奶牛乳腺炎性金葡菌诱导奶牛乳腺上皮细胞（bMECs），通过 Solexa 高通量测序，筛选 bMECs 差异性表达miRNAs；采用基因过表达或沉默法，确认免疫相关miRNAs；通过荧光素酶报告基因系统，筛选靶基因；确认 miRNAs 调控 bMECs 抗金葡菌免疫反应的信号通路；利用免疫荧光、qPCR、Western blot和ELISA，在奶牛乳腺炎细胞和动物模型中验证 miRNAs 介导信号通路对机体免疫反应的调控作用，为阐明奶牛乳腺炎性金葡菌发病机理的分子机制提供理论依据。

奶牛乳腺炎是奶牛的重大疾病之一，主要导致泌乳牛产奶量下降，降低牛奶品质和质量，给世界奶牛业带来巨大的经济损失。金黄色葡萄球菌（S. aureus）是奶牛乳腺炎最常见的病原菌之一，主要引起奶牛亚临床型和慢性临床型乳腺炎。microRNAs（miRNAs）在转录后水平调控基因表达，在生长发育、细胞增殖、分化和凋亡等一系列生命过程中发挥重要的调控作用。miRNAs 调控奶牛乳腺免疫，但它在奶牛乳腺上皮细胞抗金黄色葡萄球菌免疫反应的调控作用和分子机制不清楚。本项目采用奶牛乳腺炎病原菌金黄色葡萄球菌处理奶牛乳腺上皮细胞（bMECs），通过 Solexa 高通量测序，筛选出174个差异性表达miRNAs。以潜在候选miRNAs为研究对象，采用基因过表达或沉默方法，研究 miRNAs 在 bMECs 抗 S. aureus 免疫反应中的功能，筛选和确认bta-miR-29b为bMECs 抗 S. aureus 免疫相关靶 miRNAs。以潜在候选靶基因作为研究对象，克隆靶基因的3'-UTR 序列，构建双荧光素酶表达载体，通过双荧光素酶报告基因系统分析，筛选TRAF4为bta-miR-29b的靶基因，确认bta-miR-29b/TRAF4为bMECs 抗 S. aureus 免疫反应的信号通路，为阐明奶牛乳腺炎性金黄色葡萄球菌发病机理的分子机制提供了理论依据。