差异表达miRNAs靶向TLR4信号通路调控E.coli型奶牛乳房炎的分子机制

E.coli感染动物可启动细胞内TLR4通路，参与机体炎症反应，但其分子调控机制尚不完全清楚。申请人前期通过E.coli型乳房炎奶牛乳腺组织的全转录组测序分析，获得了4个功能未知的候选差异表达miRNAs，推测可能靶向TLR4通路基因而调控E.coli型乳房炎。因此，本研究拟采用双荧光素酶报告基因实验鉴定候选miRNA的靶基因；利用LPS分别诱导奶牛乳腺上皮细胞、单核-巨噬细胞及二者共培养细胞产生炎症反应，在此基础上，采用miRNA超表达/沉默、qPCR、WB、流式细胞等技术，揭示候选miRNA在细胞炎症反应中的表达规律，研究候选miRNAs对TLR4通路、炎症因子基因的表达、细胞增殖与凋亡的影响，探讨细胞共培养微环境对细胞炎症反应的影响，最终阐明候选miRNAs对E.coli型奶牛乳房炎的分子作用机制，为解析奶牛乳房炎的分子网络奠定基础，为乳房炎抗病分子育种提供新的理论依据。

E. coli infection can initiate intracellular TLR4 pathway and participate in inflammatory response, but its molecular regulation mechanism is not fully understood. Previously, four novel candidate differentially expressed miRNAs were obtained by whole transcriptome sequencing of the mammary gland tissues of cows with E.coli_type mastitis. It is speculated that they may target many genes of TLR4 signaling pathway to regulate E.coli_type mastitis. Therefore, this study intends to identify the target genes of candidate miRNAs by dual luciferase reporter gene assay. In addition, LPS induces mammary epithelial cells, monocytes-macrophages and co-cultured cells to produce inflammation response, respectively. On this basis, miRNAs overexpression / silencing, qPCR, Western Blot, flow cytometry and other techniques are used to reveal the dynamic expression patterns of the candidate miRNAs during the cell inflammation response, and study the effects of candidate miRNAs on expression of TLR4 pathway genes, inflammatory factor, cell proliferation and apoptosis. The results will elucidate the roles and molucular regulation mechanism of candidate miRNAs on E.coli mastitis, which will lay a foundation for understanding the molecular network of cow mastitis and provided a new theoretical basis for molecular breeding of mastitis resistance.