细粒棘球蚴原头节miR-71的分泌表达特征分析

doi:10.12140/j.issn.1000-7423.2020.02.009

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (2): 188-193.doi: 10.12140/j.issn.1000-7423.2020.02.009

细粒棘球蚴原头节miR-71的分泌表达特征分析

颜鲁军¹, 李雅婷², 丁军军³, 杨静², 郑亚东², 陈轶霞¹^,^*()

1 西北民族大学生命科学与工程学院,兰州 730030
2 中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃动物寄生虫病重点实验室,兰州 730046
3 张家川回族自治县胡川镇畜牧中心,天水 741000

收稿日期:2019-11-19 出版日期:2020-04-30 发布日期:2020-05-11
通讯作者: 陈轶霞
作者简介:颜鲁军（1994-）,男,硕士研究生,从事预防兽医学研究工作。E-mail: lujunyan1227@163.com
基金资助:
国家自然科学基金(U1703104);西北民族大学中央高校基本科研业务费创新团队培育项目(31920190027);国家重点基础研究发展计划（973计划）项目(No. 2015CB150300)

Analysis of miR-71 secretion and expression characteristics of Echinococcus granulosus protoscolex

Lu-jun YAN¹, Ya-ting LI², Jun-jun DING³, Jing YANG², Ya-dong ZHENG², Yi-xia CHEN¹^,^*()

1 Life Science and Engineering College of Northwest University for Nationalities, Lanzhou 730030, China
2 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
3 Huchuan Animal Husbandry Center, Zhangjiachuan Hui Autonomous County, Tianshui 741000, China

Received:2019-11-19 Online:2020-04-30 Published:2020-05-11
Contact: Yi-xia CHEN
Supported by:
Supported by the National Natural Science Foundation of China(U1703104);the Cultivation Project of Innovation Team for Basic Scientific Research Business Expenses of Central Universities in Northwest Minzu University(31920190027);the National Key Basic Research and Development Plan 973 Plan Project(No. 2015CB150300)

摘要/Abstract

摘要：

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。

关键词: 细粒棘球蚴, 原头节, miR-71, 表达特征, 外泌体

Abstract:

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.

Key words: Echinococcus granulosus, Protoscolex, miR-71, Expression characteristics, Exosome

中图分类号:

R383.33

颜鲁军, 李雅婷, 丁军军, 杨静, 郑亚东, 陈轶霞. 细粒棘球蚴原头节miR-71的分泌表达特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(2): 188-193.

Lu-jun YAN, Ya-ting LI, Jun-jun DING, Jing YANG, Ya-dong ZHENG, Yi-xia CHEN. Analysis of miR-71 secretion and expression characteristics of Echinococcus granulosus protoscolex[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(2): 188-193.

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细粒棘球蚴原头节miR-71的分泌表达特征分析

Analysis of miR-71 secretion and expression characteristics of Echinococcus granulosus protoscolex

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