结膜吸吮线虫外泌体miRNA在虫体与宿主互作中的功能初探

Thelaziosis is a kind of zoonosis, which is a serious public health problem in some areas of southwest China. However, knowledges of the parasite-host interactions are still limited. In recently studies, evidences proved that secreted extracellular vesicles（exosomes）play an important role during the pathogen-host interactions, in which miRNAs are transferred to host cells to play a role of regulation. Previous study confirmed that Thelazia callipaeda could secreted vesicle-like exosomes. And five highly expressed exosomal miRNAs were identified from secretion. Given that, in this research we hypothesized Thelazia callipaeda’s exosomal miRNAs may be involved in the regulation of Thelazia callipaeda-host interactions. In this study, the main composition of exosomal miRNAs secreted by Thelazia callipaeda were identified. Subsequently, intercellular transfer of parasite information to host tissues via Thelazia callipaeda exosomal miRNAs were measured by cellular biology methods combined with micro-technology. Then cytokine assay and mRNA profile assay were used to study the influence of Thelazia callipaeda exosomal miRNAs in immune response, especially that in host HCEC cells and immunocytes. Additionally, the role of miRNA tcp-let-7_R+1_1ss22TA, highly enriched in the Thelazia callipaeda based on previous study, were determined by miRNA inhibition electroporation and confocal laser scanning microscopy analyses. Our results are expected to provide new insights to further understanding the mechanism of Thelazia callipaeda-host interactions from perspective of miRNAs, which may facilitate the research to develop novel interventions for alleviating the symptom of Thelazia callipaeda as well as for preventing and treating thelaziosis.

结膜吸吮线虫病为人兽共患寄生虫病，在我国西南等地区危害严重。然而，目前对结膜吸吮线虫与宿主互作的机制认识非常有限。研究表明，分泌型外泌体（exosomes）介导了病原体与宿主的互作，其中miRNA更是被传递到宿主细胞发挥调控作用。我们前期研究发现结膜吸吮线虫分泌类似 exosomes的小囊泡，并从分泌物中鉴定出5个高表达的虫源性miRNA。为此，本研究以结膜吸吮线虫外泌体miRNA的调控功能研究为切入点，分离并鉴定结膜吸吮线虫外泌体miRNA，研究其在宿主细胞中的迁移情况和作用效应，特别是对宿主人眼结膜上皮细胞（HCEC）和免疫细胞的影响，进一步鉴定前期研究中有较多富集的虫源性miRNA tcp-let-7_R+1_1ss22TA对宿主及虫体靶向基因的调控机制。预期研究结果将从miRNA视角揭示结膜吸吮线虫与宿主互作的寄生调控机制，为探讨结膜吸吮线虫病的防治研究提供新思路。

结膜吸吮线虫病为人兽共患寄生虫病，在我国西南等地区危害严重。目前对结膜吸吮线虫与宿主互作的机制认识非常有限。研究表明，分泌型外泌体（exosomes）介导了病原体与宿主的互作，其中miRNA更是被传递到宿主细胞发挥调控作用。我们前期研究发现结膜吸吮线虫分泌类似 exosomes的小囊泡，并从分泌物中鉴定出5个高表达的虫源性miRNA。为此，本研究以结膜吸吮线虫外泌体miRNA的调控功能研究为切入点，分离并鉴定结膜吸吮线虫外泌体miRNA，研究其在宿主细胞中的迁移情况和作用效应，特别是对宿主人眼结膜上皮细胞（HCEC）和免疫细胞的影响。1.外泌体组分鉴定 利用透射电镜观察显示分离出的外泌体直径大小在50-100nm；采用质谱技术（LC-MS/MS）及组学数据鉴定出5个高表达的虫源性miRNA；2.结膜吸吮线虫miRNA调控宿主HCEC细胞研究 通过PKH67标记外泌体的表膜蛋白，将标记后的外泌体与宿主正常HCEC细胞共孵育发现虫源性外泌体miRNAs在共孵育后的宿主HCEC细胞中可以被检测到，表明结膜吸吮线虫可以携带其自身的miRNAs到宿主细胞；3.虫源性miRNA介导的免疫细胞功能研究 体外细胞研究中发现exsomes将其内在的虫源性蛋白和RNA释放至巨噬细胞，导致细胞培养基上清中的促炎症细胞因子TNF-α和IL-13的释放，表明结膜吸吮线虫exsomes可能对宿主巨噬细胞产生较强的杀伤作用；转染虫源性miRNA后，其宿主靶基因CLMP的表达下调，而TNF信号转导活性因子IL-β、IL-6、TNF-α和p38表达上调，表明Bantam可能是通过调控其靶基因的表达介导TNF信号通路的激活进而诱发细胞活性变化，抑制细胞增殖，诱导细胞凋亡。4. 虫源性miRNA介导的免疫细胞功能研究 感染结膜吸吮线虫的小鼠单核细胞和TNF-α水平升高。用miRNA模拟物或用TcpEV和TcpEV处理后注入小鼠会导致增殖，表明TcpEV miR-125b和bantam miRNA可以调节宿主通过降低Pros1，Fam212b和Clmp等基因的表达水平调控TNF-α生产，并改变巨噬细胞功能。