广州管圆线虫感染小鼠脑内差异表达microRNAs的功能及其对EOS的调控机制研究

项目名称： 广州管圆线虫感染小鼠脑内差异表达microRNAs的功能及其对EOS的调控机制研究

项目编号： No.81471980

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 李华

作者单位： 南方医科大学

项目金额： 62万元

中文摘要： 广州管圆线虫（AC）感染可导致人和小鼠嗜酸粒细胞（EOS）增多性脑膜炎或脑膜脑炎（EM），EOS主要受MMP9和细胞因子（CK）调节，其调控机制尚不明确。在其他疾病中miRNAs可调控CK的表达，我们前期研究已鉴定出AC感染小鼠脑内差异表达的miRNAs。这些miRNAs能否通过调控MMP9和CK的表达来调节小鼠脑内EOS？本项目拟①预测miRNAs与免疫调节/效应相关的靶基因，制定miR-up/down慢病毒，转染小鼠小胶质细胞，抗原刺激后检测候选靶基因、MMP9和CK的表达水平，筛选具调控功能的miRNAs及其靶基因；②双荧光素酶检测系统验证miRNA对靶基因的作用及结合位点；③miR-up/down慢病毒注射小鼠脑内，AC感染后检测miRNA与靶基因、MMP9与CK的表达水平，观察病理、EOS数量及凋亡指标变化；阐明miRNA对AC感染小鼠脑内EOS的调控机制，为EM防治提供新思路。

中文关键词： 广州管圆线虫；小鼠；MicroRNA；嗜酸粒细胞；调控机制

英文摘要： Angiostrongylus cantonensis infection can cause severe Eosinophilic meningitis (EM). MMP-9 and cytokines(ECF, IL-5, TNF-α, etc.) can regulate the EOS in EM, but the regulatory mechanism is unknown. Our previous studies have identified the differentially expressed miRNAs (18 up-regulated and 3 down-regulated ) in brain tissues of mice infected with A.cantonensis, in which miR-21, miR-223 and miR-146a are confirmed to participate in the regulation of EOS in other diseases. Can these differentially expressed miRNAs regulate EOS by regulation to MMP-9 and cytokines in brain of infected mice? This study intend to (1) Selected the forecast miRNAs with immunological regulation and effect to assemble miR-up and miR-down lentivirus and transfect into mouse microglia. To identify miRNA functions in vitro, the mRNA and protein expression levels of MMP-9 and cytokines will be detected after the A. cantonensis antigen stimulation. (2) To identify the target genes and binding sites with miRNAs, pMIR-Target-WT and pMIR-Target-MUT (pMIR-REPORT Luciferase) will be constructed according to the target genes of miRNAs validated and co-transfect murine microglia with mimics/NC. Then detect the luciferase activity.(3)To identify miRNA functions in vivo , mice will be infected with A. cantonensis after injection miR-up and miR-down lentiviruses into brain. The expression level of MMP-9 and cytokine，MMP-9 activity and changes of quantity and apoptosis of EOS will be detected 21dpi post A.cantonensis.This project will clarify the mechanism of miRNA regualtion in EM and provide a new strategy for Angiostrongyliasis control.

英文关键词： Angiostrongylus cantonensis;Mice;MicroRNA;Eosinophilic granulocyte;Regulatory mechanisms