番鸭肝细胞外泌体miRNA表达谱的研究  

Expression Profile of MicroRNA in Exosomes of Livers in Young Muscovy Duck

作　　者：赖妤婕 郑娱 游泽龙 魏祥端 许高琳 许丽惠[1] 王全溪[1] LAI Yujie;ZHENG Yu;YOU Zelong;WEI Xiangduan;XU Gaolin;XU Lihui;WANG Quanxi(College of Animal Science(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002)

机构地区：[1]福建农林大学动物科学学院(蜂学学院),福建福州350002

出　　处：《中国家禽》2023年第3期24-29,共6页China Poultry

基　　金：福建省自然科学基金(2019J01658);福建农林大学大学生创新训练项目(202110389156)。

摘　　要：为探究育雏阶段番鸭肝脏中外泌体携带miRNA的种类及其涉及的功能,试验采集3周龄雏番鸭肝脏制备原代肝细胞,培养3 d,离心取上清,试剂盒法分离肝细胞外泌体,电子显微镜观察外泌体形态和粒径,免疫印迹法检测外泌体CD81蛋白表达丰度。提取外泌体总RNA,miRNA测序后进行注释、GO和KEGG功能富集分析。结果显示:雏番鸭肝细胞外泌体为直径30~100 nm的囊泡小体,呈“杯托”状;标志性蛋白CD81免疫印迹结果呈阳性表达;miRNA测序共获得了59个新注释miRNA,GO功能富集结果主要涉及单有机体过程、刺激应答、多细胞有机体过程、定位、细胞器、胞外区绑定分子功能等;KGEE功能富集结果显示主要涉及淀粉和蔗糖代谢、MAPK信号通路、氨基糖和核苷酸糖代谢、肌醇磷酸代谢和磷脂酰肌醇信号传导系统等通路。研究表明番鸭肝脏外泌中的miRNA种类丰富,涉及多种生理功能。To explore the types and functions of micro RNAs in exosomes of livers in Muscovy duck during brooding period,the primary hepatocytes from 3-week-age Muscovy duck were prepared. The hepatocytes culturing for 3 days were collected and centrifuged. The supernatant was collected and the exosomes were isolated according to the exosome isolation kit instructions.The exosomes morphology and particle size were detected by transmission electron microscopy. The expression level of CD81 protein in the exosomes was detected by Western blot. Then the microRNAs in the exosomes were sequenced and annotated. The functions of novel miRNAs were analyzed by GO functional enrichment classification and KEGG signaling pathway functional enrichment analysis. The results showed that the diameters of exosomes from the duck hepatocytes were about 30 to 100 nm, which were "cup holder", and had a complete membrane. The marker protein CD81 was positively expressed in the exosomes detected by Western blot. Analyzing the results of the small RNA sequencing, fifty-nine novel miRNAs were annotated, which were mainly involved in single organism process, stimulus response, multicellular organism process, localization, organelle, extracellular domain binding molecular function, etc by GO. Result of KGEE signaling pathway analysis mainly focused on starch and sucrose metabolism, MAPK signaling pathway, amino sugar and nucleotide sugar metabolism, inositol phosphate metabolism and phosphatidylinositol signaling system, etc. These results indicated that the miRNAs in the liver exosomes of Muscovy duck were rich in kinds and involved in a variety of physiological functions.

关 键 词：雏番鸭 肝细胞 外泌体 小RNA 

分 类 号：S834.89[农业科学—畜牧学]