水牛犊牛血浆外泌体的分离与鉴定

doi:10.16431/j.cnki.1671-7236.2021.09.010

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (9): 3200-3205.doi: 10.16431/j.cnki.1671-7236.2021.09.010

水牛犊牛血浆外泌体的分离与鉴定

庞春英, 梁莎莎, 文崇利, 陈明棠, 陆呈委, 韦科龙, 潘玉红, 梁贤威

中国农业科学院广西水牛研究所, 农业农村部(广西)水牛遗传繁育重点实验室, 南宁 530001

修回日期:2021-04-25 出版日期:2021-09-20 发布日期:2021-09-17
通讯作者: 梁贤威 E-mail:liangbri@126.com
作者简介:庞春英(1972-),女,广西博白人,副研究员,研究方向:动物胚胎生物技术,E-mail:pangcy800@163.com
基金资助:
广西自然科学基金（2017GXNSFAA198108）；广西科技重大专项（桂科AA16450002）；国家自然科学基金项目（31660649）

Isolation and Identification of Plasma Exosomes in Buffalo Calves

PANG Chunying, LIANG Shasha, WEN Chongli, CHEN Mingtang, LU Chengwei, WEI Kelong, PAN Yuhong, LIANG Xianwei

Guangxi Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China

Revised:2021-04-25 Online:2021-09-20 Published:2021-09-17

摘要/Abstract

摘要： 研究旨在从水牛犊牛血浆中分离外泌体，并对分离的外泌体进行分子生物学特征分析和鉴定。采用差速离心法分离3头水牛犊牛的血浆外泌体，并在透射电子显微镜下观察其形态，使用纳米粒度及Zeta电位仪对提取的外泌体进行粒径大小分析，应用Western blotting方法检测外泌体标记蛋白钙联蛋白（Calnexin）、肿瘤易感基因101（TSG101）、CD9、CD81在3头水牛犊牛血浆外泌体中的表达情况。结果显示，从水牛犊牛血浆中分离的外泌体形态多为圆形和椭圆形，直径在30~150 nm；Western blotting检测结果表明，在水牛血浆外泌体表面，特异性蛋白Calnexin、TSG101和CD81阳性表达，CD9不表达。本研究从形态学和分子生物学特征等方面证实研究获得的提取物为外泌体，由此说明，差速离心法可以成功分离提取水牛犊牛血浆中的外泌体，为水牛外泌体的后续研究提供了重要技术基础和参考。

关键词: 水牛; 血浆; 外泌体; 差速离心法; 外泌体特异蛋白

Abstract: The aim of this study was to separate exosomes from the plasma of buffalo calves, analysis and identify molecular biological characteristics of the isolated exosomes. The differential centrifugation was used to separate exosomes from plasma of three buffalo calves, and the morphology of extracted exosomes was observed under a transmission electron microscope. The particle size of the extracted exosomes was analyzed using nanoparticle size and Zeta potentiometer, and the expression of exosome marked proteins, including Calnexin, tumor susceptibility gene 101 (TSG101), CD9 and CD81, were detected by Western blotting method. The results showed that plasma exosomes of buffalo calves were mostly round and elliptical, with a diameter between 30 to 150 nm. The Western blotting results showed that the specific proteins Calnexin, TSG101 and CD81 were positive expressed in the extracted plasma exosomes, but protein CD9 was negative. Based on morphology and molecular characteristics, it was confirmed that the obtained extract were exosomes, indicating that the differential centrifugation could be used to successfully isolate plasma exosomes of buffalo calves. The results provided an important technical basis and reference for further research on buffalo exosome.

Key words: buffalos; plasma; exosome; differential centrifugation; exosome specific protein

中图分类号:

Q251

庞春英, 梁莎莎, 文崇利, 陈明棠, 陆呈委, 韦科龙, 潘玉红, 梁贤威. 水牛犊牛血浆外泌体的分离与鉴定[J]. 中国畜牧兽医, 2021, 48(9): 3200-3205.

PANG Chunying, LIANG Shasha, WEN Chongli, CHEN Mingtang, LU Chengwei, WEI Kelong, PAN Yuhong, LIANG Xianwei. Isolation and Identification of Plasma Exosomes in Buffalo Calves[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3200-3205.

参考文献

[1] HE C, ZHENG S, LUO Y, et al. Exosome theranostics:Biology and translational medicine[J]. Theranostics, 2018, 8(1):237-255.
[2] TRAMS E G, LAUTER C J, SALEM N J, et al. Exfoliation of membrane ecto-enzymes in the form of micro-vesicles[J]. Biochimica et Biophysica Acta, 1981, 645(1):63-70.
[3] JOHNSTONE R M, ADAM M, HAMMOND J R, et al. Vesicle formation during reticulocyte maturation.Association of plasma membrane activities with released vesicles (exosomes)[J]. Journal of Biological Chemistry, 1987, 262(19):9412-9420.
[4] VALADI H, EKSTROM K, BOSSIOS A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells[J]. Nature Cell Biology, 2007, 9(6):654-659.
[5] PEGTEL D M, GOULD S J.Exosomes[J]. Annual Review Biochemistry, 2019, 88:487-514.
[6] SCHEY K L, LUTHER J M, ROSE K L.Proteomics characterization of exosome cargo[J]. Methods, 2015, 87:75-82.
[7] MILANE L, SINGH A, MATTHEOLABAKIS G, et al. Exosome mediated communication within the tumor microenvironment[J]. Journal of Controlled Release, 2015, 219:278-294.
[8] KAHLERT C, KALLURI R.Exosomes in tumor micro-environment influence cancer progression and metastasis[J]. Journal of Molecular Medicine, 2013, 91(4):431-437.
[9] WANG Y, BALAJI V, KANIYAPPAN S, et al. The release and trans-synaptic transmission of Tau via exosomes[J]. Molecular Neurodegeneration, 2017, 12(1):5.
[10] HAN D P, ZHU Q L, CUI J T, et al. Polo-like kinase 1 is overexpressed in colorectal cancer and participates in the migration and invasion of colorectal cancer cells[J]. Medical Science Monitor, 2012, 18(6):BR237-246.
[11] LERA R F, BURKARD M E.High mitotic activity of Polo-like kinase 1 is required for chromosome segregation and genomic integrity in human epithelial cells[J]. Journal of Biological Chemistry, 2012, 287(51):42812-42825.
[12] WANG Y, GAO C, GAO T Y, et al. Plasma exosomes from depression ameliorate inflammation-induced depressive-like behaviors via sigma-1 receptor delivery[J]. Brain Behavior and Immunity, 2021, 94:225-234.
[13] MEAD B, TOMAREV S.Bone marrow-derived mesenchymal stem cells-derived exosomes promote survival of retinal ganglion cells through miRNA-dependent mechanisms[J]. Stem Cells Translation Medicine, 2017, 6(4):1273-1285.
[14] 孙有良, 王宇童.两种提取小鼠血清来源外泌体方法的比较[J]. 中国医药导报, 2020, 17(7):4-7. SUN Y L, WANG Y T.Comparison of two different methods of extracting exosomes from mouse sera[J]. China Medical Herald, 2020, 17(7):4-7.(in Chinese)
[15] 赵翡, 朱华, 马海英, 等.BMSCs-exo对紫外线诱导成纤维细胞光老化的保护作用[J]. 解剖科学进展, 2019, 25(5):548-551. ZHAO F, ZHU H, MA H Y, et al. Protective effect of mouse bone marrow mesenchymal stem cell-derived exosomes on photoinduced aging of UV-induced fibroblasts[J]. Progress of Anatomical Science, 2019, 25(5):548-551.(in Chinese)
[16] JAVEED N, MUKHOPADHYAY D.Exosomes and their role in the micro-/macro-environment:A comprehensive review[J]. Journal of Biomedical Research, 2017, 31(5):386-394.
[17] 罗哲斯, 钟成勇, 秦转, 等.大鼠血清外泌体不同提取方法的比较[J]. 广东化工, 2019, 2(46):23-24. LUO Z S, ZHONG C Y, QIN Z, et al. Comparison and optimization of different methods for the extraction of rat serum-derived exosomes[J]. Guangdong Chemical Industry, 2019, 2(46):23-24.(in Chinese)
[18] Al-NEDAWI K, MEEHAN B, MICALLEF J, et al. Intercellular transfer of the oncogenic receptor EGFRvⅢ by microvesicles derived from tumour cells[J]. Nature Cell Biology, 2008, 10(5):619-624.
[19] LI M, LU Y, XU Y, et al. Horizontal transfer of exosomal CXCR4 promotes murine hepatocarcinoma cell migration, invasion and lymphangiogenesis[J]. Gene, 2018, 676:101-109.
[20] NAGER A R, GOLDSTEIN J S, HERRANZ-PEREZ V, et al. An actin network dispatches ciliary GPCRs into extracellular vesicles to modulate signaling[J]. Cell, 2017, 168(1-2):252-263.e14.
[21] SAMANTA S, RAJASINGH S, DROSOS N, et al. Exosomes new molecular targets of diseases[J]. Acta Pharmacologjca Sinica, 2018, 39(4):501-513.
[22] ESCOLA J M, KLEIJMEER M J, STOORVOGEL W, et al. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes[J]. Journal of Biological Chemistry, 1998, 273(32):20121-20127.
[23] HEMLER M E.Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain[J]. Annual Review of Cell and Development Biology, 2003, 19(1):397-422.

水牛犊牛血浆外泌体的分离与鉴定

Isolation and Identification of Plasma Exosomes in Buffalo Calves

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	李杨辉, 许涛, 张淑贤, 刘珂, 贾天晴, 陈宁, 尹君亮, 席琳乔, 王栋, 周小玲. 孜然秸秆替代小麦秸秆对羔羊生长和屠宰性能及血浆生化指标的影响[J]. 中国畜牧兽医, 2023, 50(6): 2296-2304.
[2]	徐媛媛, 郑海英, 杨春艳, 邓廷贤, 黄晨茜, 冯超, 陆杏蓉, 段安琴, 莫霞, 马小娅, 尚江华. 抑制BMP/Smad信号通路对水牛卵巢颗粒细胞生长和类固醇激素合成的影响[J]. 中国畜牧兽医, 2023, 50(6): 2354-2362.
[3]	肖鹏, 尚江华, 杨春艳, 李孟琪, 段安琴, 马小娅, 冯超, 黄晨茜, 张博, 周金陈, 韦科龙, 郑威, 郑海英. α-亚麻酸对水牛卵巢颗粒细胞体外培养的影响[J]. 中国畜牧兽医, 2023, 50(6): 2380-2387.
[4]	李志杰, 赵莹, 马鑫, 王萌, 余四九, 王立斌, 潘阳阳. 动物卵泡液外泌体分离方法及主要生殖功能研究进展[J]. 中国畜牧兽医, 2023, 50(4): 1480-1488.
[5]	孔维怡, 王菲菲, 岳康, 秦俊杰, 朱浩, 郭延生. 基于UPLC-MS/MS代谢组学技术分析归芪益母口服液对产后奶牛血浆代谢物的影响[J]. 中国畜牧兽医, 2023, 50(3): 913-923.
[6]	臧长江, 刘保宣, 院东, 李晓斌, 李凤鸣, 陈开旭, 米热古丽·伊马木, 杨开伦. 补喂L-精氨酸对返情纯血母马妊娠率及血浆参数的影响[J]. 中国畜牧兽医, 2023, 50(1): 125-133.
[7]	唐庆凤, 李玲, 黄加祥, 方文远, 农皓如, 唐艳, 陆呈委, 韦盘秋, 冯玲. 广西地区不同饲养模式及不同季节水牛奶霉菌毒素污染状况调查[J]. 中国畜牧兽医, 2022, 49(9): 3665-3675.
[8]	蔡萌, 朱晓艳, 王梦玲, 刘子豪, 熊本海, 杨亮. 金黄色葡萄球菌处理对奶牛乳腺上皮细胞外泌体表征的影响[J]. 中国畜牧兽医, 2022, 49(8): 3091-3098.
[9]	徐玉珍, 刘希望, 秦哲, 李世宏, 白莉霞, 范丽萍, 魏彦明, 李剑勇, 杨亚军. 液相色谱-串联质谱法检测猪血浆中除虫脲的方法学研究[J]. 中国畜牧兽医, 2022, 49(7): 2746-2756.
[10]	刘新勇, 谭正准, 李辉, 黄健, 罗华, 曾令湖, 程隽如, 钟华配, 杨春艳, 王晓丽, 覃广胜. 热应激对摩拉和尼里-拉菲水牛血液生化指标的影响[J]. 中国畜牧兽医, 2022, 49(2): 521-528.
[11]	魏文焯, 梁振华, 吴艳, 刘静波, 皮劲松, 张昊. 外泌体对动物肠道健康的影响及其研究方法[J]. 中国畜牧兽医, 2022, 49(2): 579-586.
[12]	翟亚莹, 施巧婷, 楚秋霞, 朱肖亭, 滑留帅, 张子敬, 陈付英, 祁兴磊, 王二耀, 吕世杰. 胚胎移植前供体牛与受体牛血浆外泌体miRNA差异表达分析[J]. 中国畜牧兽医, 2022, 49(2): 641-649.
[13]	徐媛媛, 莫霞, 黄晨茜, 冯超, 陆杏蓉, 马小娅, 杨春艳, 郑海英, 尚江华. 水牛TRAIL基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2022, 49(10): 3693-3703.
[14]	梁莎莎, 庞春英, 邓廷贤, 陆杏蓉, 段安琴, 马小娅, 方艳艳, 梁贤威. 水牛MFSD2A基因多态性及其与产奶性状的关联分析[J]. 中国畜牧兽医, 2022, 49(10): 3856-3865.
[15]	郑海英, 杨春艳, 李玲玉, 唐丽萍, 郑威, 尚江华. 白藜芦醇对体外培养的水牛卵丘细胞增殖活力及其相关基因mRNA表达的影响[J]. 中国畜牧兽医, 2022, 49(1): 208-215.