大熊猫脐带间充质干细胞外泌体的分离鉴定及miRNAs富集分析

doi:10.16829/j.slxb.150664

兽类学报 ›› 2023, Vol. 43 ›› Issue (1): 33-40.DOI: 10.16829/j.slxb.150664

大熊猫脐带间充质干细胞外泌体的分离鉴定及miRNAs富集分析

安俊辉^2,3, 李媛², 王东辉^2,3, 陈佳松^2,3, 李红艳², 梁小虎², 封同英², 蔡志刚^2,3, 侯蓉^2,3, 曾长军¹, 刘玉良^1,2,3

1 四川农业大学动物科技学院, 畜禽遗传资源发掘与创新利用四川省重点实验室, 成都 611130;
2 成都大熊猫繁育研究基地, 四川省濒危野生动物保护生物学重点实验室, 成都 610081;
3 四川省大熊猫科学研究院, 成都 610081

收稿日期:2022-02-25 修回日期:2022-07-05 发布日期:2023-01-10
通讯作者: 刘玉良,E-mail:sdliuyuliang@163.com
作者简介:安俊辉(1984-),男,助理研究员,主要从事濒危野生动物遗传资源保存研究;李媛(1992-),女,硕士研究生,主要从事濒危野生动物遗传资源保存研究.
基金资助:
成都大熊猫繁育研究基金会项目(CPF2017-16)；四川杰出青年科技人才项目(2020JDJQ0074)

Isolation, identification, and miRNAs enrichment analysis of exosomes derived from giant panda umbilical cord mesenchymal stem cells

AN Junhui^2,3, LI Yuan², WANG Donghui^2,3, CHEN Jiasong^2,3, LI Hongyan², LIANG Xiaohu², FENG Tongying², CAI Zhigang^2,3, HOU Rong^2,3, ZENG Changjun¹, LIU Yuliang^1,2,3

1 College of Animal Sciences and Technology, and Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Si-chuan Province, Sichuan Agricultural University, Chengdu 611130, China;
2 Chengdu Research Base of Giant Panda Breeding, Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Cheng-du 610081, China;
3 Sichuan Academy of Giant Panda, Chengdu 610081, China

Received:2022-02-25 Revised:2022-07-05 Published:2023-01-10

摘要/Abstract

摘要： 大熊猫脐带间充质干细胞(Umbilical cord mesenchymal stem cells, UC-MSCs)通过旁分泌所释放的外泌体在大熊猫保健与疾病治疗方面具有一定的应用前景。本研究旨在建立大熊猫UC-MSCs外泌体分离方法，开展生物学特征分析和分子鉴定，并研究UC-MSCs外泌体中miRNAs的种类与功能。采用超速离心法从大熊猫UCMSCs培养上清中成功分离外泌体，通过透射电子显微镜进行形态学观察，纳米颗粒跟踪分析仪检测粒径大小，蛋白免疫印迹法检测特异性分子标记表达。采用Small RNA测序技术对UC-MSCs外泌体中的miRNAs进行鉴定，并对其靶基因进行了预测与功能分析。结果显示，大熊猫UC-MSCs外泌体呈圆形杯托状结构，直径为(79.15±4.81)nm，外泌体标志蛋白CD81与TSG101呈阳性表达而CALNEXIN呈阴性表达。大熊猫UC-MSCs外泌体中的miRNA主要为miR-148-3p(30.28%)与miR-21-5p(21.72%)。本研究首次从大熊猫 UC-MSCs培养上清中分离出外泌体，并对其所含的miRNAs进行富集分析及功能预测，为大熊猫UC-MSCs外泌体后续的功能研究及应用奠定基础。

关键词: 大熊猫, 脐带间充质干细胞, 外泌体, miRNA

Abstract: The exosomes released by giant panda umbilical cord mesenchymal stem cells (UC-MSCs) through paracrine mechanisms have application prospects in giant panda veterinary care and disease treatment. The aims of this study were to explore a method of separating exosomes secreted by giant panda UC-MSCs, identify their biological and molecular characteristics, and study the miRNA expression profile and distribution characteristics. Ultracentrifugation was used to successfully isolate exosomes from the supernatant of giant panda UC-MSCs culture medium. The morphological observation was performed by transmission electron microscopy, particle size was detected by nanoparticle tracking analyzer, and the expression of specific molecular markers was detected by western blotting. In addition, the small RNA sequencing technology was used to explore the expression profile and function of exosomal miRNAs. The results showed that the exosomes obtained from giant panda UC-MSCs displayed a circular cup-shaped structure with a particle size of (79. 15 ± 4. 81) nm. These exosomes were positive for CD81 and TSG101 but negative for CALNEXIN. The miRNAs in exosomes were mainly composed of miR-148-3p (30. 28%) and miR-21-5p (21. 72%). In this study, we harvested exosomes from the supernatant of giant panda UC-MSCs culture medium for the first time and conducted the enrichment analysis and functional prediction of UC-MSCs miRNAs. This study lays the foundation for follow-up studies of UCMSCs exosomes and their clinical application in giant pandas.

Key words: Giant panda, Umbilical cord mesenchymal stem cells, Exosomes, miRNA

中图分类号:

Q341

安俊辉, 李媛, 王东辉, 陈佳松, 李红艳, 梁小虎, 封同英, 蔡志刚, 侯蓉, 曾长军, 刘玉良. 大熊猫脐带间充质干细胞外泌体的分离鉴定及miRNAs富集分析[J]. 兽类学报, 2023, 43(1): 33-40.

AN Junhui, LI Yuan, WANG Donghui, CHEN Jiasong, LI Hongyan, LIANG Xiaohu, FENG Tongying, CAI Zhigang, HOU Rong, ZENG Changjun, LIU Yuliang. Isolation, identification, and miRNAs enrichment analysis of exosomes derived from giant panda umbilical cord mesenchymal stem cells[J]. ACTA THERIOLOGICA SINICA, 2023, 43(1): 33-40.

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大熊猫脐带间充质干细胞外泌体的分离鉴定及miRNAs富集分析

Isolation, identification, and miRNAs enrichment analysis of exosomes derived from giant panda umbilical cord mesenchymal stem cells

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