目的 分离并鉴定旋毛虫新生幼虫分泌的细胞外囊泡（EV）主要成分，了解新生幼虫EV的功能。方法 消化旋毛虫保种小鼠肌肉，手机旋毛虫肌幼虫，昆明小鼠灌胃感染肌幼虫（2 000条/鼠）后5 d取小肠，网筛法收集旋毛虫成虫，成虫置于RPMI 1640培养液中培养48 h后收集新生幼虫。采用差速超速离心法提取旋毛虫新生幼虫培养液中的EV，透射电子显微镜观察EV形态，使用纳米颗粒跟踪分析技术检测EV的粒径，蛋白质免疫印迹（Western blotting）验证EV蛋白。对新生幼虫及其EV进行蛋白组质谱分析和miRNA测序分析，采用t检验比较新生幼虫及其EV之间蛋白和miRNA的表达差异。差异表达蛋白和miRNA靶基因进行基因本体论（GO）功能条目富集分析和京都基因与基因组百科全书（KEGG）通路富集分析。使用WoLFPSORT分析蛋白的亚细胞定位。结果 透射电子显微镜和纳米颗粒跟踪分析结果显示，旋毛虫新生幼虫分泌的EV为“杯盘”形膜性囊泡，平均粒径为（101.7 ± 1.8） nm。Western blotting结果显示，新生幼虫EV能与旋毛虫感染小鼠血清产生特异性条带，相对分子质量约为65 000。蛋白组质谱分析结果显示，新生幼虫及其EV样品中分别检测到1 424和231种蛋白，有220种蛋白在两组样品中均有表达，其中116种表达有差异（在EV中31种表达上调、85种表达下调）。蛋白亚细胞定位分析结果显示，差异蛋白主要分布于细胞质（30.2%，35/116）、细胞核（19.0%，22/116）和细胞膜（17.2%，20/116）。miRNA测序结果显示，新生幼虫及其EV样品分别检测到183和117种miRNA，有46种miRNA在两组样品中均有表达，其中40种表达有差异（在EV中10种表达上调、30种表达下调）。在EV中表达上调的10种miRNA中，let-7-5p表达最高。GO功能条目富集分析结果显示，差异蛋白有57种属于细胞组成、73种具有分子功能、63种参与生物过程，差异miRNA的靶基因主要与自噬相关12基因转移酶活性、胞质泛素连接酶复合物、细胞对紫外线A的响应和钙离子稳态等条目有关。KEGG通路富集分析结果显示，差异蛋白主要富集在代谢途径、内质网中的蛋白质加工等相关通路，差异miRNA的靶基因仅涉及自噬-其他1条途径。结论 本研究分离并鉴定了旋毛虫新生幼虫分泌的EV，检测到新生幼虫及其EV间差异表达的116种蛋白和40种miRNA，主要参与代谢、自噬等途径，推测新生幼虫EV可能在旋毛虫感染早期逃避宿主免疫防御的过程中发挥重要作用。

Objective To isolate and identify the main components of extracellular vesicles (EV) secreted by Trichinella spiralis newborn larvae and elucidate the function of these EVs. Methods T. spiralis muscle larvae were sourced from the muscles of mice, which had been utilized for T. spiralis preservation. The small intestine was excised 5 days after infected with muscle larvae, and T. spiralis adults were obtained through mesh screening. These adults were then cultured in RPMI 1640 medium for 48 hours to yield newborn larvae. EVs were collected by differential ultracentrifugation from newborn larvae culture fluid of T. spiralis, and were observed morphologically under transmission electron microscope, and the diameter of the newborn larvae EVs was measured by nanoparticle tracking analysis. The EV protein components were verified using Western blotting, and analyzed by proteomic mass spectrometry. RNA sequencing technology was applied to analyze the miRNA sequences of the newborn larvae and their secreted EVs. The differences of protein components and differential expression between newborn larvae and their EVs were compared using t-test. The differentially expressed proteins and miRNA target genes were analyzed by gene ontology (GO) functional item enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment. WoLFPSORT was used to analyze the subcellular localization of proteins. Results Transmission electron microscopy and nanoparticle tracking analysis revealed that T. spiralis newborn larvae produce cup-shaped, membranous vesicles with a diameter of approximately (101.7 ± 1.8) nm. Western blotting demonstrated that the newborn larvae-derived EVs generate specific bands when reacted with sera from mice infected with Trichinella spiralis, with a approximate molecular weight of 65 000. Through proteomics mass spectrometry, 1 424 and 231 proteins were detected in the newborn larvae and EV samples, respectively. Among these, 220 proteins were expressed in both groups, with 116 displaying differential expression patterns (31 upregulated and 85 downregulated in EVs). The subcellular localization analysis revealed that the differentially expressed proteins were predominantly localized in the cytoplasm (30.2%, 35/116), nucleus (19.0%, 22/116), and cell membrane (17.2%, 20/116). miRNA sequencing data showed that 183 and 117 miRNAs were detected in newborn larvae and EV samples, respectively; 46 miRNAs were found in both groups, with 40 showing differential expression patterns (10 upregulated and 30 downregulated in EVs). Notably, let-7-5p, miR-125-5p, miR-10, and miR-92 exhibited higher levels of upregulation in newborn larvae-derived EVs. The GO functional enrichment analysis indicated that the differentially expressed proteins could be categorized into three main groups: cell composition (57), molecular function (73), and biological processes (63). The target genes of differentially expressed miRNA were primarily associated with autophagy-related genes such as transferase activation of autophagy-related 12 and ubiquitin linkage enzyme complex. KEGG pathway enrichment analysis suggested that the differentially expressed proteins were significantly enriched in metabolic pathways and protein processing in endoplasmic reticulum-related pathways, whereas the target genes of differentially expressed miRNA were only involved in the autophagy-other pathway. Conclusion This study successfully isolated and identified the EVs secreted by newborn larvae of T. spiralis. 116 differentially expressed proteins and 40 miRNAs were identified between the newborn larvae and their EVs, predominantly participating in metabolic, autophagic and other pathways, which inferring that newborn larvae EVs may play important roles in evading host immune defence at early Trichinella infection.