【作者】 孙敏；

【导师】 宫鹏涛；

【作者基本信息】 吉林大学 ， 预防兽医学， 2023， 硕士

【摘要】 十二指肠贾第虫（Giardia duodenalis）,是一种单细胞人兽共患寄生性原虫,主要引起人和牛羊等多种动物的炎性腹泻,全球每年约有2.8亿人感染贾第虫。目前尚无理想防治贾第虫病的办法,因此系统阐明贾第虫与宿主间互作机制对防控贾第虫病的流行极为重要。宿主针对病原体入侵产生的先天免疫机制包括多种形式,如细胞焦亡、细胞凋亡以及细胞坏死等。细胞焦亡（Pyroptosis）是一种裂解性细胞程序性死亡,其表现为细胞不断胀大直至细胞膜破裂,细胞内容物释放出来。细胞焦亡可以通过炎症小体激活caspase-1来介导,并伴随着大量促炎症因子的释放。目前已报道的NLRP3,NLRC4和AIM2等炎症小体均可介导细胞焦亡的发生。实验室前期研究发现贾第虫能够激活宿主巨噬细胞焦亡,且贾第虫胞外囊泡（GEVs）可以通过激活MAPK、NF-κB信号通路以及NLRP3炎症小体调节宿主炎性反应。然而,贾第虫及其胞外囊泡中的何种成份参与调控宿主细胞焦亡尚未可知。贾第虫变体特异性表面蛋白（VSP）是导致贾第虫出现反复感染和慢性感染的主要原因,同时贾第虫VSP也能刺激宿主先天免疫反应。GEVs蛋白组学测序分析GEVs中存在3种VSP。本研究选择GEVs中包含的VSPAS7,通过探究其在贾第虫诱导宿主巨噬细胞焦亡中的作用及调节机制,为研究贾第虫与宿主间相互作用机制提供理论基础。主要研究包括:1.VSPAS7抑制贾第虫激活宿主NF-κB及ERK/MAPK信号通路的研究成功构建贾第虫pc DNA3.1-vspas7真核表达载体,并在小鼠巨噬细胞中表达VSPAS7。通过Western blot和ELISA分别检测贾第虫VSPAS7对贾第虫诱导巨噬细胞信号通路激活及促炎细胞因子分泌的影响。结果发现VSPAS7可以抑制贾第虫激活的p65、IκBα和ERK磷酸化,并且使贾第虫诱导的巨噬细胞促炎细胞因子IL-6、IL-12 p40和TNF-α的分泌水平下降,表明VSPAS7能够抑制贾第虫激活宿主炎症信号通路。2.VSPAS7抑制贾第虫诱导宿主巨噬细胞焦亡现象观察贾第虫刺激pc DNA3.1-vspas7真核表达质粒转染24 h后的小鼠巨噬细胞,Western blot、ELISA和LDH试验分别检测细胞焦亡关键蛋白的表达或活化、IL-1β分泌和LDH释放情况。结果发现,在贾第虫刺激细胞18 h后,VSPAS7能够使贾第虫诱导的GSDMD裂解、pro-caspase-1活化、上清中IL-1β分泌和LDH释放水平减少,表明VSPAS7抑制贾第虫对宿主细胞焦亡反应的激活。3.VSPAS7通过NLRP3/caspase-1/GSDMD抑制贾第虫诱导宿主巨噬细胞焦亡机制研究贾第虫刺激pc DNA3.1-vspas7真核表达质粒转染24 h后的NLRP3^-/-小鼠巨噬细胞18 h,Western blot、ELISA和LDH试验分别检测细胞焦亡关键蛋白的表达或活化水平、IL-1β分泌水平和LDH释放量;免疫荧光分析（IFA）、免疫共沉淀（Co-IP）和双分子荧光互补（Bi FC）试验观察贾第虫VSPAS7蛋白与小鼠NLRP3蛋白定位及互作情况。结果显示,在NLRP3^-/-小鼠细胞中,VSPAS7无法抑制贾第虫诱导的细胞GSDMD裂解、pro-caspase-1活化、上清中IL-1β分泌量和LDH释放量增加;且贾第虫VSPAS7蛋白与小鼠NLRP3蛋白可以通过直接作用的方式调节NLRP3/caspase-1/GSDMD途径,进而抑制贾第虫诱导的细胞焦亡。综上所述,VSPAS7蛋白可以抑制贾第虫激活宿主NF-κB和ERK/MAPK信号通路,降低贾第虫引起的促炎性细胞因子的分泌水平。VSPAS7蛋白可以通过与小鼠NLRP3蛋白直接作用调节NLRP3/caspase-1/GSDMD途径从而抑制贾第虫诱导的宿主巨噬细胞焦亡,本研究为贾第虫与宿主间相互作用机制提供理论基础,并为贾第虫病防治提供新的策略。更多还原

【Abstract】 Giardia duodenalis,is a single-celled parasitic protozoan that affects humans and animals,primarily causes inflammatory diarrhea in humans,cattle,sheep,and many other species,Giardia infects approximately 280 million people worldwide each year.To date,there is no ideal way to prevent and control giardiasis,and the systematic elucidation of the mechanisms of Giardia-host interactions is extremely important for the prevention and control of giardiasis.The host innate immunity to pathogenic invasion includes many forms,such as pyroptosis,apoptosis and necrosis.Pyroptosis,a lytic programmed cell death that is manifested by cell swelling until the cell membrane ruptures,resulting in the release of cellular contents.It can be mediated by the activation of caspase-1 by inflammasomes and accompanied by the release of large amounts of pro-inflammatory factors.Inflammasomes,such as NLRP3,NLRC4 and AIM2 have been reported to mediate the development of pyroptosis.Previous laboratory studies have shown that Giardia can activate host macrophage pyroptosis and that Giardia extracellular vesicles（EVs）can regulate host inflammatory responses by activating MAPK,NF-κB signalling pathways and NLRP3 inflammasome.However,the specific components of Giardia and its EVs that are involved in the regulation of host pyroptosis are still unknown.Giardia variant-specific surface protein（VSP）is the major reason of recurrent and chronic infections with Giardia.Likewise,Giardia VSP stimulates the host innate immune response.Giardia EV proteomic sequencing was revealed that three VSPs,including VSPAS7,are present in Giardia EVs.In this study,we investigated the role of VSPAS7 in Giardia-induced host macrophage pyroptosis and its regulatory mechanisms to provide a theoretical basis for the study of Giardia-host interactions.This study includes:1.VSPAS7 inhibited host NF-κB and ERK/MAPK signaling pathway activation by GiardiaThe Giardia pc DNA3.1-vspas7 eukaryotic expression vector was successfully constructed and the VSPAS7 protein was expressed in mouse macrophages.The effect of Giardia VSPAS7 protein on the activation of Giardia-induced macrophage signaling pathway and the secretion of pro-inflammatory cytokines was examined by Western blot and ELISA,respectively.The results showed that VSPAS7 protein could inhibit Giardia-activated p65,IκBαand ERK phosphorylation,and decreased the secretion levels of Giardia-induced the pro-inflammatory cytokines IL-6,IL-12 p40,and TNF-αin macrophages,indicating that VSPAS7 protein could inhibit Giardia-activated host inflammatory signalling pathways.2.VSPAS7 could inhibit Giardia-induced host macrophage pyroptosisGiardia-stimulated pc DNA3.1-vspas7 eukaryotic expression plasmid was transfected with mouse macrophages for 24 h.The expression or activation of key proteins for macrophage pyroptosis,IL-1βsecretion and LDH release were examined by Western blot,ELISA and LDH assay,respectively.The results demonstrated that the VSPAS7 protein was able to reduce the Giardia-induced GSDMD cleavage,pro-caspase-1 activation,IL-1βsecretion and LDH release levels in supernatant after18 h of Giardia stimulation of cells,indicating that VSPAS7 protein inhibited the activation of Giardia response to host pyroptosis.3.VSPAS7 inhibited Giardia-induced host macrophage pyroptosis via NLRP3/caspase-1/GSDMDNLRP3^-/-mouse macrophages were transfected with pc DNA3.1-vspas7eukaryotic expression plasmid for 24 h.After stimulation with Giardia for 18 h,Western blot,ELISA and LDH assays were performed to detect the expression or activation levels of key proteins for macrophage pyroptosis,IL-1βsecretion levels and LDH release,respectively.Immunofluorescence assay（IFA）,co-immunoprecipitation（Co-IP）and the bimolecular fluorescence complementation（BiFC）assay were performed to observe the localization and interactions between Giardia VSPAS7 and mouse NLRP3 protein.The results showed that in NLRP3^-/-mouse macrophages,VSPAS7 protein was unable to inhibit Giardia-induced cellular GSDMD cleavage,pro-caspase-1 activation,increased IL-1βsecretion in supernatant and LDH release.The NLRP3/caspase-1/GSDMD pathway can be regulated by the Giardia VSPAS7protein and mouse NLRP3 protein interactions directly,which inhibit Giardia-induced pyroptosis.In summary,VSPAS7 inhibited the Giardia-activated host NF-κB and ERK/MAPK pathways,and reduced the secretion of pro-inflammatory cytokines induced by Giardia.VSPAS7 could regulate the NLRP3/caspase-1/GSDMD pathway through direct interaction with mouse NLRP3 protein to inhibit Giardia-induced macrophage pyroptosis.This study provides a theoretical basis for the mechanism of Giardia-host interaction and a new strategy for the control of Giardiasis.更多还原