细粒棘球蚴感染宿主后诱导miRNA参与免疫应答调控的研究

In our previous studies on the recombinant vaccines of ovine, we found two recombinant vaccine candidates, which are able to stimulate immune response and protect animals from Echinococcus granulosus infection. In this proposal,we will extract miRNAs from animals before or after infection of recombinant antigens, plus with or without infection of Echinococcus larva.Then, we will conduct deep RNA sequencing and bioinformatic analysis to determine the constitution of miRNAs in these animals, compared with that from unimmunized animals that are used as control. We expect to find some miRNA candidates that are expressed at different levels in these animal groups. We will focus on the miRNAs whose expression is either increased or reduced after antigen infection. For these miRNA candidates, we will construct recombinant adenovirus with individual miRNA genes, so that they can be expressed in eukaryotic cells.The roles of these miRNAs on immune response will be studied by expressing individual miRNAs in cultured initialized lymphocytes, observing the possible direction of differentiation of lymphocytes, and monitoring the changes of related cytokines. Through this project, we expect to determine whether miRNA is involved in the host immune regulation and provide new data for the interaction between the host and the pathogen, such as the immune response and immune escape. In addition, we will test whether these miRNAs have potential diagnostic values.

在我们早期对绵羊重组疫苗免疫保护性研究中，获得了2个具有较好免疫保护力的重组疫苗候选分子，本研究拟利用重组抗原对动物免疫前后和细粒棘球蚴虫感染前后以及未经抗原免疫接种直接感染对照所获得的实验材料，提取miRNA进行测序，通过信息分析进行差异筛选，以期找出与获得免疫保护力相关的、可能参与诱导宿主机体免疫应答调节作用的miRNA分子。在此基础上利用重组真核表达体系将前期筛选可能具有调节作用的miRNA候选分子构建成腺病毒重组体，转染体外分离培养的的各种免疫细胞，观察在其参与下细胞可能的分化方向，并检测相关的细胞因子，探讨miRNA与这些因子及淋巴细胞分化间的关系。希望通过此研究观察miRNA是否参与了宿主免疫调控作用，为解决在寄生虫病研究中存在的宿主与病原体间免疫相互作用及免疫逃逸等难题提供新的资料。同时，开展包虫病患者与正常人miRNA的对照研究来探讨其是否具有诊断价值。

本研究通过制备小鼠的细粒棘球蚴重组抗原免疫模型及包虫感染模型，筛选出了重组抗原免疫前后及包虫感染前后差异表达的miRNA和LncRNA分子，将筛选出来的差异表达分子分别转染磁珠分选后的naïve CD4+ T细胞，流式细胞术检测Th 细胞亚型的分化情况，探讨miRNA及LncRNA分子与淋巴细胞分化间的关系。流式细胞术检测重组抗原免疫后小鼠脾脏及外周血中 T、B、MDSC、NK 和 NKT细胞的增殖与分化情况。结果显示，差异表达的microRNA分子一共有66个，其中上调的分子有36个，下调的分子有30个。流式细胞术结果显示，mmu-miR-126a-5p、mmu-miR-374b-5p 均可调控naïve CD4+ T 细胞向Th1方向分化；利用LncRNA 042200过表达和干扰慢病毒分别转染naïve CD4+T 细胞，结果显示，LncRNA 042200可调控naïve CD4+ T 细胞向Th1方向分化。.通过此研究探讨差异表达的miRNA及LncRNA分子是否参与了宿主免疫调控作用，为解决在寄生虫病研究中存在的宿主与病原体间免疫相互作用及免疫逃逸等难题提供新的资料。

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