目的 分析在胰岛素、阿苯达唑和人工胃液刺激下,细粒棘球蚴原头节miR-71的分泌表达特征。方法 从新疆屠宰场采集绵羊肝、肺细粒棘球蚴包囊,经固定、包埋及切片后,用miR-71探针进行杂交。与异硫氰酸荧光素（FITC）标记的抗Digoxin抗体（Anti-Digoxin-FITC）作用,经4’,6-二脒基-2-苯基吲哚（DAPI）核酸染料染色后,在荧光显微镜下观察miR-71在包囊壁和原头节中的定位,分析miR-71在原头节中的分布,确定miR-71在原头节中的表达情况。在人工胃液（人工胃液组）、胰岛素（胰岛素组）或阿苯达唑（阿苯达唑组）等不同条件下培养原头节,收集上清并用差速离心法分离外泌体,采用透射电镜观察外泌体形态结构,利用纳米粒径分析仪测定外泌体的粒径分布;利用外泌体生物标志分子烯醇化酶和14-3-3蛋白的抗体蛋白质免疫印迹（Western blotting）鉴定外泌体;采用实时荧光定量PCR（qPCR）检测外泌体中miR-71的丰度。结果 原位杂交结果显示,miR-71在囊壁生发层和原头节中均有表达。人工胃液组、胰岛素组和阿苯达唑组原头节分泌的外泌体粒径分别为48.9、49.7和65.4 nm,均在40~120 nm内;外泌体形态呈由膜包裹的球形。Western blotting从外泌体中检出烯醇化酶和14-3-3蛋白,表明外泌体提取成功。qPCR结果显示,人工胃液组、胰岛素组和阿苯达唑组原头节分泌的外泌体中,miR-71的表达量分别是其对照组的1.84、1.87和2.38倍（t = 12.8、26.7、29.3,均P < 0.01）。结论 miR-71在细粒棘球蚴囊壁和原头节中广泛表达,且可以通过外泌体进行分泌;经胰岛素、人工胃液和阿苯达唑刺激后其分泌表达水平升高。

Objective To analyze the secretion and expression characteristics of miR-71 of Echinococcus granulosus protoscolex under stimulation by insulin, albendazole and artificial gastric fluid.Methods E. granulosus cysts in sheep liver and lung tissues were collected at a slaughterhouse in Xinjiang, China. The E. granulosus cysts were fixed, embedded and sectioned, followed by hybridization with a miR-71 probe. The sections were then incubated with antibody labeled with fluorescein isothiocyanate (FITC), and then stained with a nucleic acid dye 4’, 6-diamidino-2-phenylindole (DAPI) to visualize the allocation of miR-71 in E. granulosus protoscoleces and cysts wall with fluorescence microscopy, analyzing the distribution of miR-71 in the protoscoleces at various developmental stages, for ascertaining the expression of miR-71 in the protoscoleces. In addition, the protoscoleces were cultured in the presence of insulin, albendazole and artificial gastric fluid and the supernatant was collected to isolate exosomes by differential centrifugation. The morphology of exosomes was observed under a transmission electron microscope. The particle-size distribution of the exosomes was measured by a nanoparticle size analyzer. The enolase and 14-3-3 protein were used as biomarkers for identification of exosome by Western blotting. The abundance of miR-71 in exosomes was determined by real-time quantitative PCR (qPCR).Results In situ hybridization showed that miR-71 was expressed in the middle and late developmental stages of protoscoleces and in the germinal layer of cyst wall of E. granulosus. The exosomes had a size of 48.9 nm, 49.7 nm and 65.4 nm in the groups of artificial gastric fluid, insulin, and albendazole, respectively, all within the range of 40-120 nm, and morphologically shaped as a membrane-enveloped sphere. Western blotting detected the presence of enolase and 14-3-3 protein in exosomes, demonstrating successful extraction of exosomes. qPCR showed that the expression level of miR-71 in exosomes secreted from protoscoleces in the test group with artificial gastric fluid, insulin, and albendazole was 1.84, 1.87 and 2.38 folds of the control group (t = 12.8, 26.7 and 29.3, P < 0.01), respectively.Conclusion miR-71 is widely expressed in protoscoleces and cyst wall of E. granulosus, and could be secreted via exosomes. The expression and secretion of miR-71 can be upregulated by stimulations with insulin, artificial gastric fluid and albendazole.