差速超速离心法与试剂盒法提取娟姗牛乳源性外泌体的分析比较

范士杰; 马露; 卜登攀

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

动物营养学报 >

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

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

研究简报 SHORT COMMUNICATION

差速超速离心法与试剂盒法提取娟姗牛乳源性外泌体的分析比较

范士杰 ,
马露 ,
卜登攀

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1. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193;
2. 中国农业科学院与世界农用林业中心农用林业与可持续畜牧业联合实验室, 北京 100193

范士杰(1992-),男,河南鲁山人,硕士研究生,研究方向为养殖。E-mail:m18811527116@163.com

收稿日期: 2020-03-24

网络出版日期: 2020-11-16

基金资助

国家重点研发计划（2018YFD0501600）；中国农业科学院科技创新工程（ASTIP-IAS07）；北京市奶牛产业创新团队（BAIC06-2018）；畜产品安全工程安徽省重点实验室开放基金（No.xm2001）

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

FAN Shijie ,
MA Lu ,
BU Dengpan

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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

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摘要

本试验旨在探究不同方法提取娟姗牛乳源性外泌体的优缺点。采用差速超速离心法和试剂盒法2种提取方法对娟姗牛乳源性外泌体进行提取，通过透射电镜（TEM）、Bradford蛋白定量、免疫印迹（WB）和粒径分析（NTA）方法对2种提取方法得到的外泌体进行比较鉴定，并采用细胞毒性试验对试剂盒法提取的外泌体进行细胞毒性评估。结果显示：2种提取方法均获得了茶托样结构的囊泡，但TEM视野下试剂盒法提取的外泌体较差速超速离心法多。试剂盒法提取的外泌体蛋白浓度显著高于差速超速离心法（P<0.05）。2种提取方法均能够检测到外泌体的标志性蛋白凋亡连接基因2相互作用蛋白X （ALIX）和四次跨膜蛋白（CD81）；差速超速离心法提取的外泌体CD81蛋白丰度极低，且并未鉴定出标志性蛋白肿瘤敏感基因101蛋白（TSG101），但试剂盒法检测到了TSG101。2种提取方法得到的外泌体粒径分布均符合外泌体的正常粒径大小（30~150 nm）。进一步通过细胞毒性试验发现，试剂盒法提取的外泌体对细胞的增殖分化几乎没有影响。因此，基于本试验条件下，试剂盒法更适用于提取娟姗牛乳源性外泌体。

关键词： 外泌体; 娟姗牛乳; 差速超速离心法; 试剂盒法

本文引用格式

范士杰 , 马露 , 卜登攀 . 差速超速离心法与试剂盒法提取娟姗牛乳源性外泌体的分析比较[J]. 动物营养学报, 2020 , 32(11) : 5441 -5447 . DOI: 10.3969/j.issn.1006-267x.2020.11.051

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

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