let-7a/g/f差异表达和并联协同靶向调控奶牛乳腺炎炎症介质IL-6的分子机制

Many biological mechanisms are still urgent to be revealed in the “black box” of cow mastitis pathogenesis. Based on the research progress at home and abroad and preliminary work, we have proposed a hypothesis that the differentially expressed let-7a/g/f can participate in the cow mastitis inflammatory response by targeting and regulating inflammatory mediator IL-6 in parallel and synergistic manner. Next, we will carry out the following experiments to confirm it. Firstly, the qualitative and quantitative analysis of let-7a/g/f in the breast tissue of healthy and S. aureus infected mastitis cow will be investigated respectively by using in situ hybridization and fluorescence quantitative PCR technology. Meanwhile, differential expression of relevant genes involved in IL-6/STAT3 and NF-kB signal pathway will be detected via IL-6/STAT3 signal pathway PCR Chip. Secondly, the regulation function of let-7a/g/f targeting IL–6 is to be verified through dual luciferase report system, and building the model of heat inactivated S. aureus induced mammary epithelial cells inflammation, then mammary gland epithelial cells apoptosis and gene expression of related pathways will be investigated by over-expression, inhibiting expression, fluorescence quantitative PCR and flow cytometry. Lastly, the regulation law of combinated transcription factors and differentially methylated region in promoter region on let-7a/g/f transcription will also be revealed by utilizing bioinformatics, dual luciferase report gene, point mutations, electrophoretic mobility shift assay (EMSA) and methylation process.

奶牛乳腺炎发病机制“黑匣子”中仍有许多生物学机制亟待揭秘。基于国内外研究进展和前期工作，我们提出“差异表达的let-7a/g/f通过对炎症介质IL-6并联协同靶向调控参与奶牛乳腺炎症反应”的假设并实验求证。首先，利用原位杂交和Q-PCR对健康和患病奶牛乳腺组织中的let-7a/g/f进行定性和定量分析，用IL-6/STAT3信号转导通路PCR芯片检测IL-6/STAT3和NF-kB信号通路中相关基因的差异表达；其次，用双荧光素酶报告基因验证let-7a/g/f对IL-6的靶向调控，并建立热灭火金黄色葡萄球菌感染的乳腺上皮细胞模型，采用基因过表达、抑制表达、Q-PCR和流式细胞等技术研究乳腺上皮细胞的凋亡以及通路中相关基因的表达规律；最后，利用双荧光素酶报告基因、点突变、EMSA和甲基化处理等方法揭示启动子区域关键转录因子和差异甲基化对let-7a/g/f转录的调控规律。

奶牛乳腺炎发病原因“黑匣子”中仍有许多生物学机制亟待揭秘。基于国内外研究进展和前期工作，我们提出“差异表达的let-7a/g/f通过对炎症介质IL-6并联协同靶向调控参与奶牛乳腺炎症反应”的假设并实验求证。研究内容及结果如下：.1.原位杂交实验检测了let-7a在健康和炎症乳腺中的组织定位与表达，发现let-7a定位于细胞胞质，且患病组织显著低于正常乳腺组织，RT-qPCR实验表明Let-7a/g/f在患病乳腺组织中表达量均显著降低，而IL-6以及相关基因smad3、KLF4、MAP3K7则与之相反；.2.利用LPS和LTA诱导，经ELISA和实时无标记检测方法成功建立体外奶牛乳腺炎上皮细胞炎症模型，确定了LPS和LTA的最佳处理时间和浓度；.3.合成了let-7a/g/f 的mimics和inhibitor，完成了IL-6 3’UTR表达载体的构建，通过细胞培养、共转染及双荧光素酶活性检测，确定Let-7a、let-7g、let-7f均可靶向调控IL-6的表达；.4.进行了Let-7a/g/f干扰实验。利用细胞分组转染、Q-PCR、ELISA、流式细胞术、RTCA检测等方法完成了Let-7a/g/f在乳腺上皮细胞炎症反应过程中的功能分析，发现过表达Let-7a/g/f均可显著降低IL-6的转录和蛋白表达水平，抑制乳腺上皮细胞的凋亡，促进其增殖；.5.在生物信息学预测Let-7a/f启动子基础上，构建了不同大小的截短性片段载体，通过细胞分组转染、双荧光素酶活性检测，确定了Let-7a/f的核心启动子区域为g.-1109～g.+43，且在g.-2176～-2152区域存在一个25bp片段缺失，经生物信息学预测，该位点存在多个转录因子；.6.构建了CCR7 3’UTR表达载体，通过细胞培养、共转染、双荧光素酶活性检测，确定Let-7a可靶向调控CCR7的表达，抑制奶牛乳腺上皮细胞的凋亡，促进乳腺上皮细胞的增殖；.7. IL-6相关通路关键基因CCL28筛选出三个SNP（g.C-601G，g.T72G和g.A14819G），经PCR-RFLP分型、单倍型组合以及关联分析，发现H3H3为最佳抗性单倍型，H3H2为最易致病单倍型。.总之，该研究初步解析了let-7a/g/f靶向调控炎症介质 IL-6 的分子机制及其功能，筛选的分子标记也为奶牛乳腺炎的抗性选育提供了一个途径。