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

项目名称： 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

项目编号： No.31460670

项目类型： 地区科学基金项目

立项/批准年度： 2015

项目学科： 水产学、兽医学

项目作者： 杜丽

作者单位： 海南大学

项目金额： 48万元

中文摘要： 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。

中文关键词： 羊种布鲁氏菌；巨噬细胞；白细胞分化抗原14；微小RNA；作用机制

英文摘要： 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.

英文关键词： B. melitensis;macrophage;CD14;miRNAs;action mechanism