多房棘球蚴囊泡来源EVs及其emu-miR-10-5p促进小鼠肝星状细胞活化  

Echinococcus multilocularis metacestode vesicle-derived EVs containing emu-miR-10-5p promote mouse hepatic stellate cell activation

作　　者：王宇琦 达哇卓玛 刘川川[1,2,3] 樊海宁 WANG Yu-qi;DAWA Zhuo-ma;LIU Chuan-chuan;FAN Hai-ning(School of Clinical Medicine,Qinghai University,Xining 810001,China;Qinghai University Affiliated Hospital,Xining 810001,China;Qinghai Province Research Key Laboratory for Echinococcosis,Xining 810001,China)

机构地区：[1]青海大学临床医学院,西宁810001 [2]青海大学附属医院,西宁810001 [3]青海省包虫病研究重点实验室,西宁810001

出　　处：《中国人兽共患病学报》2024年第5期454-463,482,共11页Chinese Journal of Zoonoses

基　　金：青海省科技厅项目(No.2023-ZJ-992Q);青海省“昆仑英才·高端创新创业人才”项目。

摘　　要：目的明确多房棘球绦虫囊泡来源胞外囊泡(Extracellular vesicles,EVs)及其emu-miR-10-5p在体外调节HSCs活化中的作用。方法小鼠腹腔注射原头节构建继发性多泡型包虫病模型,以小鼠体内病灶为材料培养多房棘球绦虫囊泡并进行PCR鉴定。透射电镜观察培养囊泡组织中胞外囊泡结构。差速-超速离心法分离囊泡培养上清中EVs,透射电镜、纳米粒径以及Western blot对分离EVs进行鉴定。CCK-8检测不同浓度EVs作用后HSCs活力。EVs作用于HSCs后RT-qPCR和Western blot检测α-SMA、CollagenⅠ和CollagenⅢ表达水平。生物信息学分析确定emu-miR-10-5p为特异性miRNA。过表达emu-miR-10-5p后EdU检测HSCs增殖,RT-qPCR个Western blot检测细胞中α-SMA、CollagenⅠ和CollagenⅢ表达水平。干扰囊泡组织中emu-miR-10-5p后提取培养上清液中EVs作用于HSCs后,检测HSCs细胞中α-SMA、CollagenⅠ和CollagenⅢ表达水平。结果成功通过小鼠体内病灶材料培养出多房棘球蚴囊泡,经PCR扩增特异性基因证实为多房棘球绦虫来源。TEM观察培养囊泡表面有较多的囊状结构。通过差速-超速离心分离出多房棘球蚴囊泡来源EVs,并经TEM证实为球形、膜状结构。囊泡来源EVs能被HSCs内化,并促进HSCs增殖和活化。通过综合分析多房棘球绦虫感染小鼠血清、棘球蚴组织、棘球蚴囊泡来源EVs、生发层细胞、原头节miRNA测序结果,发现emu-miR-10-5p在上述成分中均存在表达,并通过RT-qPCR证实emu-miR-10-5p在多房棘球蚴囊泡来源EVs中存在的量较高。进一步研究证实过表达emu-miR-10-5p能促进HSCs增殖,增加HSCs中α-SMA、Collagen I和CollagenⅢmRNA及蛋白表达水平。而干扰囊泡中emu-miR-10-5p后从培养上清液中提取的EVs未增加α-SMA、Collagen I和CollagenⅢmRNA及蛋白表达水平。结论多房棘球蚴囊泡来源EVs及emu-miR-10-5p能够促进HSCs活化。This study investigated the role of Echinococcus multilocularis vesicle-derived extracellular vesicles(EVs) containing emu-miR-10-5p in regulating HSC activation in vitro.Mice were injected intraperitoneally with protoscoleces to construct a secondary alveolar echinococcosis model,and E.multilocularis vesicles were cultured in mice and identified by PCR.The EV structure in cultured vesicle tissue was observed by transmission electron microscopy.EVs were isolated from vesicle culture supernatants through differential-ultracentrifugation,and identified by transmission electron microscopy,nanoparticle size analysis,and western blotting.The activity of HSCs treated with different EV concentrations was detected with CCK-8 assays.The expression levels of α-SMA,and Collagen Ⅰ and Ⅲ were detected by RT-qPCR and western blotting after EV treatment of HSCs.Bioinformatics analysis identified the specific miRNA emu-miR-10-5p.The proliferation of HSCs was detected with EdU after overexpression of emu-miR-10-5p,and the expression levels of α-SMA,Collagen Ⅰ,and collagen Ⅲ in cells were detected by RT-qPCR and western blotting.Detection of α-SMA,Collagen Ⅰ,and collagen Ⅲ expression levels in HSCs was performed after EVs were extracted from the culture supernatant of vesicular tissues with emu-miR-10-5p knockdown.E.multilocularis vesicles were successfully cultured from focal material in mice,and the E.multilocularis origin was confirmed through PCR amplification of specific genes.TEM observation of culture vesicles indicated abundant surface vesicular structures.E.multilocularis vesicle-derived EVs were isolated by differential-ultracentrifugation,and confirmed by TEM to be spherical and membrane like-structures.Vesicle-derived EVs were internalized by HSCs,and promoted the proliferation and activation of HSCs.Comprehensive analysis of miRNA sequencing results revealed emu-miR-10-5p expression in E.multilocularis-infected mouse serum,metacestode tissue,E.multilocularis-derived EVs,germinal layer cells,and protosco

关 键 词：多房棘球蚴病 胞外囊泡 肝星状细胞 emu-miR-10-5p 

分 类 号：R383.3[医药卫生—医学寄生虫学]