一种大容量细胞灌流液中外泌体的提取方法及鉴定*

doi:10.12047/j.cjap.5989.2020.111

中国应用生理学杂志 ›› 2020, Vol. 36 ›› Issue (5): 524-528.doi: 10.12047/j.cjap.5989.2020.111

• 技术方法 • 上一篇

一种大容量细胞灌流液中外泌体的提取方法及鉴定^*

李兰兰¹, 聂文青², 贺燕婷¹, 王美月¹, 李振凤³, 李宏¹, 官秀梅¹, 成敏^△

1.潍坊医学院基础医学院, 2.潍坊市妇幼保健院, 3.潍坊医学院医学研究中心, 山东潍坊 261053

收稿日期:2019-12-18 修回日期:2020-06-19 发布日期:2021-02-25
通讯作者: ^△Tel: 0536-8462268; E-mail: mincheng@wfmc.edu.cn
基金资助:
^*国家自然科学基金资助项目(81870237,31570941,81700406); 山东省高校科研计划项目(J14LK12,J18KA275); 潍坊医学院大学生科技创新课题(KX2019081);潍坊医学院公派国内访学项目经费资助

A method of exosomes extraction from large-volume cell perfusate

LI Lan-lan¹, NIE Wen-qing², HE Yan-ting¹, WANG Mei-yue¹, LI Zhen-feng³, LI Hong¹, GUAN Xiu-mei¹, CHENG Min^1△

1. Basic Medical School of Weifang Medical University, 2. Weifang Maternal and Child Health Hospital, 3. Medicine Research Center of Weifang Medical University, Weifang Medical University , Weifang 261053, China

Received:2019-12-18 Revised:2020-06-19 Published:2021-02-25

摘要/Abstract

摘要： 目的: 建立一种从大容量细胞灌流液中提取外泌体的方法,并进行外泌体的鉴定。方法: 人脐静脉内皮细胞株(EA.HY926)是人脐静脉内皮细胞和人肺腺癌细胞株A549杂交成的永生化细胞株,因其具有血管内皮细胞的特性,广泛用于内皮细胞相关研究。本研究采用含10%胎牛血清的M199培养基培养,利用Flexcell STR-4000平行板流室系统对EA.HY926施以振荡剪切应力。收集流体剪切应力处理后的细胞灌流液,去除细胞碎片后冻成干粉,脱盐、提取纯化外泌体。电镜观察外泌体形态、纳米粒径电位分析仪检测外泌体大小、PKH26染色检测外泌体膜性结构、BCA蛋白定量法检测外泌体的蛋白浓度、Western blot检测外泌体特异性蛋白CD9和CD81的表达,荧光定量RT-PCR检测内皮细胞相关基因的表达。结果: 该方法提取的外泌体,大小均一,结构完整,呈典型囊泡样结构;粒径集中在30~150 nm,多数粒径为97.63 nm;表达外泌体特异性蛋白CD9和CD81;PKH26染色阳性,并可被细胞摄取;EA.HY926分泌的外泌体表达内皮细胞相关的CD31、vWF等mRNA,以及miR-126、miR-21、miR-155等microRNA分子。结论: 本方法能够有效从大容量细胞灌流液中提取到结构完整的、高浓度、高质量的外泌体,为开展以流体力学干预细胞为基础的外泌体相关研究提供技术方法。

关键词: 外泌体, 细胞灌流液, 流体剪切应力, 人脐静脉内皮细胞株

Abstract: Objective: To establish an efficient method for extracting exosomes from large-volume cell perfusate. Methods: EA.HY926, an immortalized cell line produced by the hybridization of human umbilical vein endothelial cells and human lung adenocarcinoma cell line A549, was cultured with M199 culture medium containing 10% fetal bovine serum. Flexcell STR-4000 parallel plate flow chamber system was employed to apply shear stress to EA.HY926. And then the perfusate was collected. The cell debris was removed by centrifugation. The supernatant was freeze-dried into the dry powder and was resuspended by small-volume medium. The dialysis was used to desalt and purify the suspension. The exoEasy Maxi Kit was used to extract the exosomes. The morphology of exosomes was observed by electron microscopy. The size of exosomes was detected by nanometer particle size analyzer. The activity of exosomes was detected by PKH26 staining. BCA protein quantification method was used to detect the protein concentration of exosomes. The expressions of exosomal specific proteins CD9 and CD81 were detected by Western blot. The quantitative RT-PCR was used to detect the expression of related genes in the exosomes. Results: The exosomes extracted by this method were uniform in size, showing a typical and complete vesicle-like structure. The particle size was concentrated at 30～150 nm, and the peak value was at 97.63 nm, indicating that the size was appropriate and the purity was high. Moreover, exosomes-specific protein CD9 and CD81 were expressed. PKH26 could bind to the membranous structure of exosomes and exosomes could be efficiently taken up by cells. Endothelial cells-associated CD31, vWF mRNA, and microRNA molecules such as miR-126, miR-21, miR-155 were expressed in exosomes secreted by EA.HY926. Conclusion: This method can effectively extract structurally intact, high-concentration, high-quality exosomes from large-volume cell perfusate.

Key words: exosomes, cell perfusate, shear stress, human umbilical vein endothelial cell line

中图分类号:

R543.1

李兰兰, 聂文青, 贺燕婷, 王美月, 李振凤, 李宏, 官秀梅, 成敏. 一种大容量细胞灌流液中外泌体的提取方法及鉴定^*[J]. 中国应用生理学杂志, 2020, 36(5): 524-528.

LI Lan-lan, NIE Wen-qing, HE Yan-ting, WANG Mei-yue, LI Zhen-feng, LI Hong, GUAN Xiu-mei, CHENG Min. A method of exosomes extraction from large-volume cell perfusate[J]. CJAP, 2020, 36(5): 524-528.

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一种大容量细胞灌流液中外泌体的提取方法及鉴定^*

A method of exosomes extraction from large-volume cell perfusate

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