日本血吸虫exosomes介导虫体与宿主互作的功能初探

Dioecious flatworms of the genus Schistosoma cause schistosomiasis, which is a major public health problem in developing countries. In China, schistosomiasis japonica is a serious public health problem. Knowledge of the parasite-host interaction are still limited. In recent studies, evidences proved that exosomes play important roles in signal transduction and regulation in cells. Moreover, in our recent work, five schistosoma -specific miRNAs were identified in the plasma of rabbits infected with S. japonicum. Further studies proved that these Schistosoma-specific miRNAs with high abundance were also observed in exosomes secreted by S. japonicum. Given this research, we hypothesized that schistosome exosomes may be involved in the regulation of schistosome-host interaction. In this study, the main composition of exosomes secreted by S. japonicum will be identified. Subsequently, intercellular transfer of parasite information via S. japonicum exosomes will be measured by cellular biology methods combined with microtechnology. Then cytokine assay and mRNA profile assay will be used to study the influence of S. japonicum exosomes in immune response. The functions of exosomal miRNAs on their targets in macrophages will be further analyzed by luciferase assay. Our results are expected to provide new insights for further understanding the mechanisms of schistosome-host interaction that may facilitate in the development of novel interventions for alleviating the symptom of S. japonicum infection as well as for preventing and treating schistosomiasis.

日本血吸虫病人兽共患，危害严重。然而，目前有关血吸虫与宿主互作的机制认识仍非常有限。最近研究发现细胞释放的外泌体（exosomes）在细胞间信号传递和调控中发挥重要作用。我们前期研究发现感染日本血吸虫的宿主血浆中存在着虫体特异性的miRNAs分子，且这些分子大量存在于分离到的日本血吸虫exosomes中。因此，我们推测日本血吸虫分泌的exosomes可能参与血吸虫与宿主互作的寄生调控。为此，本研究拟从日本血吸虫exosomes的调控功能研究为切入点，分离并鉴定日本血吸虫exosomes中的主要组份，研究虫源性exosomes在细胞中迁移情况和作用效应，特别是对宿主巨噬细胞的免疫因子及mRNA表达谱变化的影响，进一步鉴定虫源性exosomal miRNAs对宿主靶向基因的调控机制。预期研究结果将从exosomes视角揭示日本血吸虫与宿主互作的寄生调控机制，为探讨血吸虫病的防治手段提供新思路。

分泌型的外泌体（exosomes）在病原体与宿主互作中发挥重要的调控作用。为此，本研究以日本血吸虫exosomes的调控功能研究为切入点，从exosomes视角揭示日本血吸虫与宿主互作的寄生调控机制，为探讨血吸虫病的防治手段提供新思路。首先，结合透析、超滤、超速离心和商用抽提试剂盒，筛选日本血吸虫exosomes的分离和纯化方法；利用透射电镜观察exosomes的形态；运用质谱、测序以及生物信息学技术研究exosomes中的蛋白和RNA组份。其次，将虫源性exosomes与巨噬细胞共孵育，荧光分别标记虫源性exosomal蛋白和RNA，荧光显微镜观察exosomal 蛋白和RNA在巨噬细胞中的迁移情况；运用ELISA及qRT-PCR等技术检测宿主免疫细胞因子表达变化和虫源性miRNAs的表达，结合测序技术研究共孵育后宿主细胞mRNA表达谱变化；进一步，建立小鼠过敏反应模型，将虫源性的exosomes注射进入小鼠体内，研究虫源性exsomes对小鼠免疫应答的调控作用。最后，生物信息学方法预测虫源性exosomal miRNA Bantam在宿主中的潜在靶基因，检测其在感染日本血吸虫小鼠组织中的表达，利用荧光素酶方法验证靶基因。体外转染Bantam mimics至巨噬细胞，研究Bantam对细胞增殖和凋亡的影响及其潜在调控途径。结果显示分离出的日本血吸虫exosomes直径大小在50-100nm。质谱结果共鉴定出403个蛋白，这些蛋白主要参与结合、催化、翻译调节等过程。Small RNA测序鉴定出了15个已知的虫源性miRNAs(cut-off reads >100)，另有约1,284,966 reads (3.4%, 2,540个unique small RNAs)能与宿主基因组匹配，其中包括一个宿主源性的miRNA ocu-miR-191-5p(4,548 reads)，表明日本血吸虫exosomes中既包括虫源性的遗传信息也包含宿主源性的信息，其可能参与虫体发育及血吸虫致病过程。此外，虫源性exosomes可以被巨噬细胞吞噬，释放虫源性exosomal蛋白和RNA至巨噬细胞，且刺激细胞因子IL-13, TNF 和 IL-5的释放，调控宿主Th2型免疫应答。筛选到3个潜在Bantam宿主靶基因CLMP、FAM212B和PRTG，其在感染日本血吸虫的宿主组织中下调。