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

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.

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。

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增多与MMP9、细胞因子（CK）等相关，但机制不明。本研究建立AC感染小鼠模型，从感染小鼠脑内发现21个差异性表达miRNAs，通过靶基因预测，从体外和体内能验证，阐明miRNA在AC感染导致EM中的作用及其机制。取得了以下研究结果：.1.从AC感染小鼠脑组织中筛选鉴定了25个差异性表达的miRNAs（24个上调和1个下调）。验证发现小鼠小胶质细胞受AC抗原刺激后mmu-miR-146a-5p表达升高，并通过其靶基因Traf6调节TNF-α的表达。.2.发现AC幼虫排泄分泌性蛋白（ESP）可抑制小鼠小胶质细胞增值，并证实mmu-miR-181a-5p通过Bcl-2/Bax抑制由AC-ESP诱导的小胶质细胞凋亡。.3.经体内外验证证实AC感染及AC-ESP刺激均引起mmu-miR-155表达升高，而mmu-miR-155通过靶蛋白SOCS1经STAT3信号通路调控MMP9，经NF-KB信号通路调控TLSP表达，从而调控小鼠EOS水平。我们发现AC感染能明显导致外周血中EOS的浸润，而敲低miR-155后，AC感染小鼠外周血EOS的数量又明显减少；与此同时，NF-KB和STAT3通路能正反馈调控miR-155的表达。上述结论有效解释了AC感染引起小鼠EOS升高的机制以及广州管圆线虫病的自限性。.4.发现蜈蚣可作为AC的宿主传播广州管圆线虫病，引起广泛关注。.5.用蛋白质组学分析和表征了大鼠和小鼠脑内AC幼虫的差异表达蛋白，29差异表达种蛋白质，并发现dim-1和act-1与不同宿主中广州管圆线虫的发育和致病性有关。.6.在寄生虫药物作用机制方面，发现氟苯达唑能下调Bcl-2和Bcl-XL的表达，而上调活化型PARP-1，caspase 3和caspase 9的表达，导致胶质瘤细胞发生G2/M期阻滞，通过激活内源性凋亡途径诱导胶质瘤细胞凋亡。乙胺嘧啶能下调Bcl-2的表达，而上调活化型caspase 3的表达，通过激活内源性凋亡途径诱导前列腺癌细胞凋亡。.发表SCI论文4，核心期刊论文2篇，统计源1篇，另2篇SCI期刊论文已完成准备投稿；会议论文8篇，特邀报告1次，参编教材/专著2部；参加学术交流10次，培养博士生2名、硕士生3名；与境外2位专家建立了良好的合作关系。