羊种布鲁氏菌感染条件下巨噬细胞受CD14影响的miRNAs的鉴定及其作用机制研究

Brucellosis is an extremely important zoonotic bacterial infectious disease. In our previous study, CD14 knockdown was shown to effectively inhibit B. melitensis M5-stimulated TNFα release and NO production in RAW264.7 cell;the microarray data of miRNAs expressing profiling of CD14 knockdown RAW264.7 cell （224.3）stimulated with B. melitensis M5 indicated that fifteen miRNAs were differentially expressed. In order to further analyze the function of CD14 in the process that B.meliltensis affect the miRNAs expression profile in macrophage and cause inflammation reaction, based on these findings, the differentially expressed miRNAs are confirmed by qRT-PCR; several bioinformatics softwares are used to predict their target genes; 3'UTR report gene experiments and pull-down experiments are performed to confirm the binding between miRNAs and mRNAs; mimics and inhibitors experiments are performed to analyze its functions. These findings will be very important for elucidating the regulation mechanism of Brucella-CD14-miRNAs-target genes -inflammation reaction and provide the important basis for the prophylaxis and therapy of Brucellosis.

布鲁氏菌病是一种重要的人兽共患传染病。在前期的工作中发现，在B. melitensis M5 感染条件下, CD14 knockdown 的RAW264.7细胞（224.3）的TNFα分泌和NO产生减少；15条miRNAs差异表达(芯片筛选结果)。为了从miRNAs的层面深入探讨羊种布鲁氏菌感染条件下CD14影响宿主炎症反应的分子机制，以之为基础，本课题拟应用qRT-PCR对芯片筛选结果进行验证；应用PicTar, TargetScan 等软件预测其靶基因；应用3'UTR荧光素酶报告基因实验和pull-down 实验对miRNA和mRNA之间的互作进行结构学验证；应用mimics过表达或inhibitor抑制miRNA表达实验对miRNA和mRNA之间的互作进行功能学验证, 从而阐释布鲁氏菌-CD14-miRNAs-靶基因-炎症反应的调控机制，为布鲁氏菌病的防治提供重要依据。

围绕着布鲁氏菌的致病机制的阐释开展了以下研究。其结果对于布病防治具有重要意义。..1. 阐释了在布鲁氏菌感染条件下，CD14对于巨噬细胞miRNAs表达的影响机制.基于前期工作，分析了B. melitensis M5-90感染CD14 knock down RAW264.7后的miRNA表达谱。结果发现，CD14 knock down显著上调mmu-miR-199a-3p 和 mmu-miR-183-5p的表达，其靶基因涉及免疫反应等。在其104条靶基因中，qRT-PCR 和 Western blot证明了Cbl-b的下调。.2. 阐释了omp25在布鲁氏菌感染巨噬细胞，影响宿主细胞miRNAs及mRNAs表达的影响.构建了B. melitensis M5-90 omp25缺失株。分析了其感染RAW264.7后的miRNA表达谱。结果发现，8条miRNA差异表达，qRT-PCR验证了mmu-miR-146-a-5p 和mmu-miR-155-5p的上调和mmu-miR-149-3p 和mmu-miR-5126 的下调。分析了mRNA表达谱。结果发现，967个基因差异表达。miRNA–mRNA 联合分析结果表明：17个基因是mmu-miR-149-3p 的靶基因。qRT-PCR验证了9个基因的差异表达。.3. 分析了LPS刺激下巨噬细胞miR-27a-5p、TDAG51的作用机制.基于前期工作，研究发现：miR-27a-5p预测靶基因-MCPIP1 mRNA表达在E.coli LPS刺激后升高。萤光素酶报告基因实验提示：miR-27a-5p可以与MCPIP1 3’UTR特异性结合。在E.coli LPS和B.melitensis LPS刺激条件下，MCPIP1过表达可显著降低RAW264.7 IL-6，IL-10和IL-1β的分泌。.在LPS刺激下，在TDAG51 Knockdown 的RAW264.7, 细胞扩增和细胞周期进程发生显著变化。.4. 克隆和表达了B.melitensis M5-90的两个重要功能基因.开展了B.melitensis M5-90 LpxK、KdtA 基因的克隆和原核表达工作。