Comparison of Jersey Milk Derived Exosomes Extracted by Differential Ultracentrifugation Method and Kit Extraction Method

FAN Shijie; MA Lu; BU Dengpan

doi:10.3969/j.issn.1006-267x.2020.11.051

2020 , Vol. 32 >Issue 11: 5441 - 5447

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.11.051

Comparison of Jersey Milk Derived Exosomes Extracted by Differential Ultracentrifugation Method and Kit Extraction Method

FAN Shijie ,
MA Lu ,
BU Dengpan

Expand

1. State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. Chinese Academy of Agricultural Sciences and World Agroforestry Center Joint Lab on Agroforestry and Sustainable Animal Husbandry, Beijing 100193, China

Received date: 2020-03-24

Online published: 2020-11-16

Fold

Abstract

The aim of this study was to explore the advantages and disadvantages between two different methods used for extracting exosomes from Jersey milk. Exosomes was firstly extracted by differential ultracentrifugation method and kit extraction method, and then the exosomes was compared and identified by transmission electron microscopy (TEM), Bradford protein quantitative, western blot (WB) and nanoparticle tracking analysis (NTA) methods, and the cytotoxicity test was performed for exosomes extracted by kit extraction method. The results showed that the two extract methods obtained the typical saucer-like structure vesicles, but under the TEM field of vision, the exosomes extracted by kit extraction method were more than those extracted by differential ultracentrifugationmethod. The concentration of exosomes protein extracted by kit extraction method was significantly higher than that extracted by differential ultracentrifugation method (P<0.05). The exosomes marker protein apoptosis-linked gene-2 interacting protein X (ALIX) and transmembrane protein (CD81) were detected by two extract methods; the protein abundance of CD81 extracted by differential ultracentrifugation method was particularly low, and the tumor susceptibility gene 101 protein (TSG101) was not identified, but the TSG101 was identified by kit extraction method. The particle size distribution of exosomes obtained from the two extract methods was consistent with the normal size of exosomes (30 to 150 nm). Further cytotoxicity test proved that the exosomes extracted by kit extraction method had no disadvantage on the proliferation of cell. In conclusions, kit extraction method is a suitable strategy for exosomes extracted from Jersey milk based on this experimental condition.

Key words： exosomes; Jersey milk; differential ultracentrifugation method; kit extraction method

Cite this article

FAN Shijie , MA Lu , BU Dengpan . Comparison of Jersey Milk Derived Exosomes Extracted by Differential Ultracentrifugation Method and Kit Extraction Method[J]. Chinese Journal of Animal Nutrition, 2020 , 32(11) : 5441 -5447 . DOI: 10.3969/j.issn.1006-267x.2020.11.051

References

[1] GURUNATHAN S,KANG M H,JEYARAJ M,et al.Review of the Isolation,characterization,biological function,and multifarious therapeutic approaches of exosomes[J].Cells,2019,8(4):307.
[2] TKACH M,THÉRY C.Communication by extracellular vesicles:where we are and where we need to go[J].Cell,2016,164(6):1226-1232.

[3] SKOTLAND T,SANDVIG K,LLORENTE A.Lipids in exosomes:current knowledge and the way forward[J].Progress in Lipid Research,2017,66:30-41.
[4] KALLURI R,LEBLEU V S.Discovery of double-stranded genomic DNA in circulating exosomes[J].Cold Spring Harbor Symposia on Quantitative Biology,2016,81:275-280.
[5] HORIBE S,TANAHASHI T,KAWAUCHI S,et al.Mechanism of recipient cell-dependent differences in exosome uptake[J].BMC Cancer,2018,18:47.
[6] ANDERSON J D,JOHANSSON H J,GRAHAM C S,et al.Comprehensive proteomic analysis of mesenchymal stem cell exosomes reveals modulation of angiogenesis via nuclear factor-kappa B signaling[J].Stem Cells,2016,34(3):601-613.

[7] SAMUEL M,CHISANGA D,LIEM M,et al.Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth[J].Scientific Reports,2017,7:5933.
[8] BADAWY A A,EL-MAGD M A,ALSADRAH S A.Therapeutic effect of camel milk and its exosomes on MCF7 cells in vitro and in vivo[J].Integrative Cancer Therapies,2018,17(4):1235-1246.

[9] WATSON D C,BAYIK D,SRIVATSAN A,et al.Efficient production and enhanced tumor delivery of engineered extracellular vesicles[J].Biomaterials,2016,105:195-205.
[10] SOMIYA M,YOSHIOKA Y,OCHIYA T.Biocompatibility of highly purified bovine milk-derived extracellular vesicles[J].Journal of Extracellular Vesicles,2018,7(1):1440132.
[11] MIYAKE H,LEE C,CHUSILP S,et al.Human breast milk exosomes attenuate intestinal damage[J].Pediatric Surgery International,2020,36(2):155-163.

[12] BENMOUSSA A,LEE C H C,LAFFONT B,et al.Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions[J].The Journal of Nutrition,2016,146(11):2206-2215.

[13] GAO H N,GUO H Y,ZHANG H,et al.Yak-milk-derived exosomes promote proliferation of intestinal epithelial cells in an hypoxic environment[J].Journal of Dairy Science,2018,102(2):985-996.
[14] AGRAWAL A K,AQIL F,JEYABALAN J,et al.Milk-derived exosomes for oral delivery of paclitaxel[J].Nanomedicine:Nanotechnology,Biology,and Medicine,2017,13(5):1627-1636.

[15] IZUMI H,TSUDA M,SATO Y,et al.Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages[J].Journal of Dairy Science,2015,98(5):2920-2933.

[16] LOBB R,MÖLLER A.Size exclusion chromatography:a simple and reliable method for exosome purification[M]//KUO W,JIA S.Extracellular vesicles.New York,NY:Humana Press,2017,1660:105-110.
[17] ZERINGER E,BARTA T,LI M,et al.Strategies for isolation of exosomes[J].Cold Spring Harbor Symposia on Quantitative Biology,2015,2015(4):319-323.
[18] 汪翔.娟姗牛的杂交应用研究进展[J].中国奶牛,2016(9):12-19.
[19] 梁霄,刘鹭,张书文,等.不同品种原料乳理化特性分析[J].食品科学,2013,34(5):50-54.
[20] 王洋,于静,王巍,等.娟姗牛品种特性及适应性饲养研究[J].中国奶牛,2011(11):47-48.
[21] ASADI J,FERGUSON S,RAJA H,et al.Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals[J].Micron,2017,99:40-48.
[22] VASWANI K,KOH Y Q,ALMUGHLLIQ F B,et al.A method for the isolation and enrichment of purified bovine milk exosomes[J].Reproductive Biology,2017,17(4):341-348.

[23] HOCK A,MIYAKE H,LI B,et al.Breast milk-derived exosomes promote intestinal epithelial cell growth[J].Journal of Pediatric Surgery,2017,52(5):755-759.

[24] REINHARDT T A,LIPPOLIS J D,NONNECKE B J,et al.Bovine milk exosome proteome[J].Journal of Proteomics,2012,75(5):1486-1492.

[25] LÖTVALL J,HILL A F,HOCHBERG F,et al.Minimal experimental requirements for definition of extracellular vesicles and their functions:a position statement from the International Society for extracellular vesicles[J].Journal of Extracellular Vesicles,2014,3:26913.
[26] YU S R,ZHAO Z H,SUN L M,et al.Fermentation results in quantitative changes in milk-derived exosomes and different effects on cell growth and survival[J].Journal of Agricultural and Food Chemistry,2017,65(6):1220-1228.

[27] LIAO Y L,DU X G,LI J,et al.Human milk exosomes and their microRNAs survive digestion in vitro and are taken up by human intestinal cells[J].Molecular Nutrition & Food Research,2017,61(11):1700082.

Options

Outlines

模态框（Modal）标题

Abstract

Cite this article

References