旋毛虫DNase II在虫体肠道寄生中突破嗜酸性粒细胞防御的机理研究

The scientific proposition of Trichinella spiralis (T. spiralis) defeat eosinophils-mediated immune responses in intestinal was determined to illuminate the the pathogenesis of T. spiralis. The ability of nuclease to degrade DNA, a scaffold and a major component of extracellular traps (ETs), is the most effective way to abolish the novel microbicidal mechanism. However, it is still unclear whether the phenomenon exists in T. spiralis with 125 DNase II-like family proteins. The work in our laboratory has showed previously that the excretion-secretion products in T. spiralis displayed the degradation activity of DNA. In our study, the active DNase II of the excretion-secretion products in T. spiralis at different stage of intestinal will be screened by nuclease zymography combined with nano-LC-ESI-MS/MS. Eosinophils from rats co-incubated with T. spiralis in vitro and rats infected with T. spiralis in vivo will be undertaken to establish model for parasite infection. The DNase II activity was blocked by the DNase II inhibitor, antibody for DNase II, and RNAi. The formation and characteristic of eosinophils ETs will be investigated in vitro and in vivo by immunofluorescence technique, scanning electron microscopy, immunohistochemical staining, and proteomics. And eosinophils ETs-mediated parasite killing will be determined by an inverted microscope. This study will illuminate the mechanism of the evasion of eosinophils-mediated immune responses in T. spiralis infection during intestinal stage.

虫体如何突破肠道嗜酸性粒细胞屏障作用是揭示旋毛虫致病机理首要解决的科学命题。核酸酶降解固有免疫细胞胞外捕获器(extracellular traps, ETs)的骨架DNA被认为是病原体逃避这种新型防御机制最有效途径。而旋毛虫125种庞大的DNase II家族蛋白是否是虫体突破宿主ETs防御的关键尚不清楚。鉴于申请人前期试验证实了旋毛虫排泄分泌产物具有降解DNA的活性，本研究拟采用酶谱结合质谱技术筛选和验证旋毛虫寄生肠道不同时期排泄分泌产物的DNase II，以旋毛虫体外感染大鼠嗜酸性粒细胞和体内感染大鼠为模型，利用DNase II抑制剂、抗体和RNA干扰技术封闭旋毛虫DNase II，采用免疫荧光、扫描电子显微镜、免疫组化、活体成像和蛋白组学等技术从体外和体内研究嗜酸性粒细胞ETs的形成情况，并分析嗜酸性粒细胞ETs介导的体内外杀虫作用，旨在阐明旋毛虫突破肠道嗜酸性粒细胞防御的机理。

核酸酶降解固有免疫细胞胞外诱捕网（extracellular traps, ETs）的骨架DNA被认为是病原体逃避这种新型防御机制最有效途径。而旋毛虫125种庞大的DNase II家族蛋白是否是虫体突破宿主ETs防御的关键尚不清楚。本项目以旋毛虫感染嗜酸性粒细胞为研究对象，以DNase II降解嗜酸性粒细胞胞外诱捕网（eosinophil extracellular traps, EETs）为突破点，初步明确了DNase II在旋毛虫肠道寄生中突破嗜酸性粒细胞防御中的作用，通过本项目的研究，主要取得了以下研究进展：.（1）旋毛虫寄生肠道不同时期排泄分泌产物具有核酸酶活性。.（2）旋毛虫重组Tsp_06558蛋白具有降解DNA的核酸酶活性，同时对革兰氏阳性菌和阴性菌生物被膜具有一定的抑制和清除作用。.（3）siRNA 368-390干扰Tsp_06558对旋毛虫虫体的存活以及虫体排泄分泌产物的核酸酶活性无明显影响。.（4）活的虫体无法激发嗜酸性粒细胞释放EETs，但灭活的旋毛虫、封闭DNase II和干扰旋毛虫Tsp_06558可刺激嗜酸性粒细胞EETs形成。.（5）封闭DNase II或干扰旋毛虫Tsp_06558对嗜酸性粒细胞杀伤虫体无作用，但孵育4 h以上对新生幼虫具有一定的杀虫作用。.（6）旋毛虫感染大鼠肠道嗜酸性粒细胞增多，封闭DNase II或干扰旋毛虫Tsp_06558不影响旋毛虫感染大鼠肠道嗜酸性粒细胞的数量以及旋毛虫肠道期肌幼虫荷虫率、成虫荷虫率和成虫产虫率，但明显降低肌肉期肌幼虫荷虫率。.本项目基本完成了原计划内容，明确了排泄分泌产物具有降解DNA的核酸酶活性，且Tsp_06558降解EETs有助于新生幼虫逃避杀伤。