差异超速离心法对不同种属间充质干细胞外泌体分离的可靠性研究

doi:10.13418/j.issn.1001-165x.2022.3.09

中国临床解剖学杂志 ›› 2022, Vol. 40 ›› Issue (3): 292-296.doi: 10.13418/j.issn.1001-165x.2022.3.09

差异超速离心法对不同种属间充质干细胞外泌体分离的可靠性研究

赵仁礼^1,2，　赖国华¹，　吴家昌¹，　吴铭杰¹，　庄伟达¹，　欧阳钧³*，　桑宏勋^1,2*

1.南方医科大学深圳医院骨科，深圳 518100；　 2. 南方医科大学第三临床医学院，广州 510630
3.南方医科大学基础医学院人体解剖学教研室广东省医学生物力学重点实验室，广州 510515

收稿日期:2021-05-31 出版日期:2022-05-25 发布日期:2022-05-31
通讯作者: 欧阳钧，教授，E-mail: jouyang@126.com；桑宏勋，主任医师，E-mail: hxsang@smu.edu.cn
作者简介:赵仁礼（1992-），男，河北人，在读硕士，研究方向：骨免疫、移植免疫耐受，E-mail: renlzhao@163.com
基金资助:
国家自然科学基金（81672180）；三名工程深圳市医疗项目（SZSM20162019）；深圳市数字外科3D打印重点实验室(ZDSYS201707311542415)；深圳市基础研究重点项目（JCYJ20200 109150641992）；广东省重点领域研发计划资助（2020B010165004）

Study on the reliability of differential hypervelocity centrifugation for isolation of mesenchymal stem cells derived exosomes from different species

Zhao Renli^1,2, Lai Guohua¹, Wu Jiachang¹, Wu Mingjie¹, Zhuang Weida¹, Ouyang Jun³*, Sang Hongxun^1,2*

1. Department of Orthopedics, Shenzhen Hospital of Southern Medical University, Shenzhen 518100, China; 2. The Third Clinical Medical College of Southern Medical University, Guangzhou 510630, China; 3. Department of Human Anatomy, Guangdong Provincial Key Laboratory of Medical Biomechanics, Southern Medical University, Guangzhou 510515, China

Received:2021-05-31 Online:2022-05-25 Published:2022-05-31

摘要/Abstract

摘要： 目的　分离培养大鼠及小鼠骨髓间充质干细胞(BMSCs)，通过差异超速离心法分离纯化外泌体并鉴定其形态及生物学性质，分析此方法对外泌体分离的可靠性。方法将培养3-5代的骨髓间充质干细胞进行成骨、成脂、成软骨诱导分化并进行染色，同时鉴定细胞表面标志物；通过透射电镜、纳米颗粒示踪分析及Western Blot鉴定分离纯化的外泌体。结果通过对成骨、成脂、成软骨分化后染色，大鼠及小鼠BMSCs具备干细胞特有的多向分化潜能。流式检测结果显示大鼠和小鼠BMSCs表达干细胞标志性表面抗原；透射电镜下，小鼠及大鼠BMSCs外泌体呈典型双层膜的杯托结构；平均粒径大小为107 nm和152 nm；Western Blot结果显示其表达外泌体标志性蛋白。结论我们能成功分离培养大鼠及小鼠的骨髓间充质干细胞；同时，差异超速离心法能够可靠地分离纯化出大鼠及小鼠骨髓间充质干细胞来源的外泌体。

关键词: 骨髓间充质干细胞, 　外泌体, 　差异超速离心法

Abstract: Objective　To isolate and culture the mouse and rat bone marrow mesenchymal stem cells (BMSCs)， purify the exosome by differential hypervelocity centrifugation， identify the morphology and biological function of exosomes and analyze the reliability of this method for exosome isolation.　Methods　BMSCs of 3-5 passages were differentiated by osteogenic, adipogenic and chondrogenic induction and then stained. The surface markers of BMSCs were identified by flow cytometry. Exosomes were identified by transmission electron microscopy, nanoparticle tracer analysis and Western Blot. Results Stained after osteogenic, adipogenic and chondrogenic induction, BMSCs were proved to have the unique potential of multidirectional differentiation. Flow cytometry results showed that mouse and rat BMSCs expressed the surface antigen of stem cells. Under transmission electron microscopy, exosomes showed a typical goblet structure with an average particle size of 107 nm and 152 nm. Western Blot results showed that the exosomes expressed landmark proteins.　Conclusions　Mouse and rat BMSCs are successfully isolated and cultured. Meanwhile, differential hypervelocity centrifugation is suitable and reliable to isolate and purify exosomes from BMSCs.

Key words: Bone marrow mesenchymal stem cells, 　Exosomes;　Differential hypervelocity centrifugation

中图分类号:

Q249

赵仁礼, 赖国华, 吴家昌, 吴铭杰, 庄伟达, 欧阳钧, 桑宏勋. 差异超速离心法对不同种属间充质干细胞外泌体分离的可靠性研究[J]. 中国临床解剖学杂志, 2022, 40(3): 292-296.

Zhao Renli, Lai Guohua, Wu Jiachang, Wu Mingjie, Zhuang Weida, Ouyang Jun, Sang Hongxun. Study on the reliability of differential hypervelocity centrifugation for isolation of mesenchymal stem cells derived exosomes from different species[J]. Chinese Journal of Clinical Anatomy, 2022, 40(3): 292-296.

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差异超速离心法对不同种属间充质干细胞外泌体分离的可靠性研究

Study on the reliability of differential hypervelocity centrifugation for isolation of mesenchymal stem cells derived exosomes from different species

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