多房棘球蚴外泌体的分离鉴定及免疫调节作用研究

Hydatid disease is an important endemic zoonotic disease caused by echinococcus tapeworm in humans and animals, which seriously harms human health and economic development in Qinghai-Tibet Plateau. Alveolar hydatid cyst is characterized as infiltrative growth, extremely poor prognosis and high fatality rate, so it is known as "insect cancer". Exosomes are nano-sized vesicles released from many kinds of cells, which contain protein and RNA and play important roles in promoting cell-to-cell communications. Deep researches on exosomes are conducive to illuminate the pathogenesis of metastasis and immunoregulatory effect.. Balb/c mice infected with Echinococcus multilocularis were used as animal model in this project. Exosomes were obtained through in vitro culture of protoscoleces and high-speed gradient centrifugation, and identified by electron microscopy, flow cytometry and nanosight tracker. Then expression profiling of microRNAs derived from exosomes were analyzed by Solexa high-throughput sequencing, and microRNAs with crucial function were predicted by bioinformatics methods. And the immunological activity of exosomes were studied in vivo and in vitro. This research will contribute to understand the relationship of parasite-host interactions.

包虫病是由棘球属绦虫寄生人和动物引起的具有地方流行特征的重要人兽共患寄生虫病，严重危害青藏高原人类健康和经济发展。多房棘球蚴呈浸润性生长，预后极差，病死率极高，素有“虫癌”之称。外泌体是由多种细胞分泌的囊泡小体，其中含有蛋白质和RNA等活性组分，具有重要的细胞信号传递功能。深入研究外泌体有助于阐明多房棘球蚴浸润转移及免疫调控等致病机制。本项目拟以多房棘球绦虫感染Balb/c小鼠作为动物模型，通过原头蚴体外培养技术和高速梯度离心获得多房棘球蚴外泌体，利用电子显微镜、流式细胞仪、Nanosight等技术对其生物学特征进行鉴定；采用Solexa高通量测序技术对外泌体中microRNAs进行分析，利用生物信息学方法预测具有重要生物学功能的microRNA；通过体内外实验，研究外泌体的免疫学活性。本项目的开展，将为阐明多房棘球蚴与宿主的相互作用奠定基础。

多房棘球蚴病是由多房棘球绦虫幼虫感染导致的严重危害人体健康和畜牧业发展寄生虫病，随着健康中国重要规划的推进，包虫病已经被列为重点防治的疾病。外泌体可作为细胞间通讯介体将miRNA输送至靶细胞，进而发挥免疫调控、细胞功能调节、影响疾病进程等重要生物学作用。外泌体在寄生虫致病机制方面已成为研究热点。本课题以多房棘球绦虫外泌体作为切入点，采用梯度超速离心方法从原头蚴体外培养液中获得了虫源性外泌体，利用透射电子显微镜成像技术和纳米流式检测技术对外泌体基本生物学特征进行鉴定，其粒径主要分布在55-130nm之间，峰值为75nm，平均粒径为86nm，外泌体浓度为4.99×109/mL，透射电子显微镜成像可见典型的圆形囊泡结构，直径约为100nm。通过Solexa高通量测序技术分析了外泌体miRNA的表达情况，结合生物信息学和分子生物学，初步探讨了一部分外泌体miRNA对巨噬细胞的作用，发现虫源性miRNA能够下调促炎性细胞因子（IL-1α、IL-6、TNF-α）的表达和上调抑炎因子IL-4，以及诱导巨噬细胞RAW264.7向M2型极化，外泌体调控作用具有双重性，其免疫调节作用有利于形成虫体与宿主之间的免疫平衡状态，使得虫体不易被宿主完全免疫消除并持续寄生。本课题初步探讨了外泌体中miRNA对巨噬细胞的免疫调节作用，为阐明多房棘球蚴与宿主的相互作用提供实验依据，对深入了解多房棘球绦虫的免疫调节机制具有一定创新意义。