CsCP介导肝吸虫感染早期免疫逃逸的分子机制研究

Many studies have shown that cysteine proteases (CPs) are closely related to the early stage of parasite infection, such as nutritional uptake, tissue invasion, immune escape and other parasitic processes. A study has confirmed that CP of Clonorchis sinensis (CsCP) can participate in metacercariae excystment as an endogenous key enzyme. In our previous work, we found that CsCP was transcribed and expressed throughout the whole life cycle of Clonorchis sinensis and was significantly up-regulated in the excysted juvenilestage compared with the metacercariae stage; recombinant CsCP (rCsCP) has good immunogenicity and reactivity; the anti-rCsCP serum incubation had a certain effect on the activity of metacercariae. The above results suggested that CsCP is a multifunctional protein, and its up-regulation in the excysted juvenile stage may be a strategy for adapting to host environment and avoiding being attacked in the early stage of parasite infection. In recent years, the infection rate of Clonorchis sinensis has been increasing year by year in many endemic areas including Guangxi. Through in vitro and in vivo experiments, this project intends to further explore the role and molecular mechanism of CsCP in the host invasion and immune escape of Clonorchis sinensis early infection by using transcriptomics, proteomics and other methods, which will provide experimental basis for elucidating the mechanism of early infection and parasitism of Clonorchis sinensis, as well as new ideas and potential targets for the prevention and treatment of clonorchiosis.

众多研究显示，半胱氨酸蛋白酶类（CPs）与寄生虫感染早期的营养摄取、组织入侵、免疫逃逸等寄生过程密切相关。研究证实，肝吸虫CP（CsCP）可作为内源性关键酶参与囊蚴脱包囊。我们的前期工作发现CsCP在肝吸虫的整个生活史均有转录表达，且后尾蚴阶段相对囊蚴阶段有明显上调；重组CsCP（rCsCP）具有良好的免疫原性和反应性；anti-rCsCP血清孵育对囊蚴的活力有一定影响。以上提示，CsCP是一种多功能蛋白，其在后尾蚴阶段上调可能是虫体感染早期为适应宿主环境、躲避攻击所采取的策略。近年来，肝吸虫的感染率在包括广西在内的多个流行区仍呈现逐年增长的趋势。本项目拟通过体内外实验，采用转录组学、蛋白质组学等手段，深入探究CsCP在肝吸虫感染早期入侵宿主、免疫逃逸等方面发挥的作用及其分子机制，为阐明肝吸虫早期感染和寄生机理提供实验基础，也为肝吸虫病的预防和治疗提供新思路和潜在靶点。

肝吸虫病依然是全球性不可忽视的人畜共患病，给流行区造成严重的社会经济负担。肝吸虫可长期寄生于宿主的肝胆管内，引发多种肝胆疾病。然而，目前关于肝吸虫幼虫在入侵早期的调控策略所知甚少。本研究旨在动态研究肝吸虫感染早期对小鼠的全身和肝脏的影响，并对比研究感染不同阶段小鼠的疾病状态。组织病理染色结果显示，肝吸虫感染后，BALB/c小鼠的肝脏病变是动态的，先逐步加重，5-10周最为严重，之后逐渐减轻。血液指标测定结果表明，在感染早期主要诱发宿主产生CRP、IL-6等指标升高的急性炎症反应，到中晚期（5周及以后）转变以Th2为主的抑炎性免疫反应（IL-6和IL-1β降低，IL-4和IL-10升高）。多组学联合分析结果表明肝吸虫幼虫入侵早期（7天及以内）会刺激肝脏富集解毒、先天性免疫（NLR及TLR）、炎症（TNF及MAPK）、物质代谢、肿瘤（p53和PI3K-Akt）及生长调节（甲状腺/类固醇激素）等信号通路，这些通路与肝吸虫的致病和虫体的生长发育密切相关。与此同时，间隙/紧密连接、ECM-受体相互作用及细胞周期等信号通路也显著性富集，这可能与肝纤维化及胆管上皮增生等发生密切相关。肠道转录组分析显示肝吸虫感染早期会显著影响小鼠肠道的昼夜节律、体液免疫及物质代谢等基因的表达。囊蚴及培养不同时间点后尾蚴的转录组数据显示，后尾蚴一方面会通过调控间隙连接及溶酶体等基因表达来实现入侵和附着，另一方面会通过调控成核、物质代谢、生长激素、背腹轴形成等基因来促进虫体自身的生长发育。此外，从囊蚴到后尾蚴检测到丰富的组织蛋白酶基因的表达变化。肝吸虫半胱氨酸蛋白酶（CsCP）属于Cathepsin L，表达于生活史各阶段，尤其是成虫阶段。我们的研究表明rCsCP对于小鼠结肠炎有较好的治疗效果，其主要机制为上调Treg/Th2细胞因子及下调结肠IL-12等促炎基因。此外，口服免疫及储存实验表明B.s-CsCP重组芽孢具有免疫原性好、促进肠道健康、安全及易储存等优势，是很有前途的防治肝吸虫病的口服疫苗。本项目研究不仅从分子层级揭示了肝吸虫与宿主早期互作的机制，为肝吸虫病的早期干预和治疗提供了新视角，还为肝吸虫虫源性蛋白制剂应用于防治肝吸虫病以及炎症性疾病提供了新思路。