试剂盒法与差速超速离心法所提血清外泌体的形态学比较
| 作者: |
1王鑫伟,
1毛建文
1 广东药科大学，广东省生物活性药物研究重点实验室，广州 510006 |
| 通讯: |
毛建文
Email: jianwenmao@hotmail.com |
| DOI: | 10.3978/j.issn.2095-6959.2016.11.029 |
| 基金: | 国家自然科学基金面上项目, 31371144 |
摘要
目的：比较常用的外泌体提取方法，获得可靠有效的血清外泌体提取方法。方法：电镜下观察鉴定差速超速离心法、试剂盒法及组合优化方法所提外泌体的形态；蛋白印迹法检测外泌体标志蛋白CD63的表达。结果：差速超速离心法提取的外泌体呈茶托样双层膜结构，直径100 nm左右；试剂盒法提取的外泌体颗粒多，大小50~200 nm之间，无明显的立体结构，经差速超速离心法纯化后形态与差速超速离心法类似，经试剂盒法纯化后与仅使用一次试剂盒提取的外泌体无明显形态差异；四种方法所提外泌体表达标志蛋白CD63。结论：差速超速离心法是更为可靠且有效的外泌体提取方法。
关键词：
外泌体
血清
差速超速离心法
试剂盒法
The morphological comparison of serum exosome extracting by ultracentrifugation and ExoQuick kit
CorrespondingAuthor: MAO Jianwen Email: jianwenmao@hotmail.com
DOI: 10.3978/j.issn.2095-6959.2016.11.029
Abstract
Objective: To evaluate the reliability and efficiency of different methods for serum-derived exosomes extraction. Methods: Electron microscope was used to identify exosomes extracted from rat serum by ultracentrifugation, ExoQuick reagent or combinatorial optimization methods. The expression of exosomal marker protein CD63 was detected by Western blotting. Results: Exosomes isolated from ultracentrifugation exerted a saucer-like bilayer membrane structure, the diameter was around 100 nm. More particles were detected using ExoQuick reagent. However, these particles did not have apparent three-dimensional structure, the diameter was between 50~200 nm. The result obtained by using ultracentrifugation after ExoQuick regent treatment was the same with independently using ultracentrifugation. There was also no significant difference between using two cycles of ExoQuick regent treatment and using it only once. CD63 was expressed in exosomes isolated by using the all four methods. Conclusion: Ultracentrifugation is a more reliable and efficient method for serum-derived exosomes extraction.