牙周膜干细胞来源外泌体的生物学特性分析

doi:10.19439/j.sjos.2019.04.002

上海口腔医学 ›› 2019, Vol. 28 ›› Issue (4): 343-348.doi: 10.19439/j.sjos.2019.04.002

牙周膜干细胞来源外泌体的生物学特性分析

赵力如^1,2, 毛家奇^1,2, 赵丙姣^1,3, 陈静^1,3

1.复旦大学附属口腔医院口腔生物医学工程实验室,上海 200001;
2.河北医科大学口腔医院,河北石家庄 050017;
3.复旦大学附属口腔医院正畸科,上海 200001

收稿日期:2019-01-24 修回日期:2019-03-12 出版日期:2019-08-25 发布日期:2019-09-23
通讯作者: 赵丙姣,E-mail：joyce_zhao_ortho@fudan.edu.cn
作者简介:赵力如（1993-）,女,硕士,住院医师,E-mail:13393317041@163.com
基金资助:
国家自然科学基金（81701011）; 上海市青年科技英才扬帆计划项目（17YF1416500）

Isolation and biological characteristics of exosomes derived from periodontal ligament stem cells

ZHAO Li-ru^1,2, MAO Jia-qi^1,2, ZHAO Bing-jiao^1,3, CHEN Jing^1,3

1. Oral Biomedical Engineering Laboratory, Shanghai Stomatological Hospital, Fudan University. Shanghai 200001;
2. Hospital of Stomatology, Hebei Medical University. Shijiazhuang 050017, Hebei Province;
3. Department of Orthodontics, Shanghai Stomatological Hospital, Fudan University. Shanghai 200001, China

Received:2019-01-24 Revised:2019-03-12 Online:2019-08-25 Published:2019-09-23

摘要/Abstract

摘要： 目的采用超速离心,分离牙周膜干细胞（periodontal ligament stem cells, PDLSCs）来源的外泌体,检测其生物学特性。方法采用有限稀释法,体外分离、培养人PDLSCs。收集PDLSCs培养上清,采用梯度离心法分离、纯化外泌体。对PDLSCs来源的外泌体进行检测,透射电镜下观察外泌体形态;蛋白质印迹法检测外泌体表面标志CD9、CD63、CD81和TSG101的表达;纳米颗粒跟踪分析（nanosight tracing analysis,NTA）检测PDLSCs来源外泌体的产量及其粒径分布特征。结果成功分离出PDLSCs来源的外泌体。透射电镜下可见,外泌体为碟形或椭圆形膜性结构,中央电子密度较低。免疫印迹结果显示,分离得到的囊泡表达外泌体特异性标志物CD9、CD63、CD81和TSG101。NTA显示,PDLSCs来源的外泌体浓度为（3.80±0.39）×10⁸颗粒/mL,粒径分布峰值在（119±12.1）nm。结论采用梯度超速离心法可以得到PDLSCs来源的外泌体,其具有特征性的外泌体膜蛋白成分和形态特征。

关键词: 牙周膜干细胞, 外泌体, 胞外囊泡, 超速离心

Abstract: PURPOSE: To isolate and identify exosomes derived from periodontal ligament stem cells (PDLSCs) collected by ultracentrifugation. METHODS: Using the limiting dilution technique, human PDLSCs were isolated and expanded. The cell culture supernatant of PDLSCs was collected. Exosomes were collected and purified with a ultracentrifugation method. Biological characteristics of exosomes derived from PDLSCs were measured by transmission electron microscopy (TEM), Western blot and nanosight tracing analysis (NTA). RESULTS: Exosomes could be successfully isolated from the supernatant of PDLSCs by a ultracentrifugation method. Under TEM, the PDLSC-derived exosomes exhibited elliptic or saucer-like shape and the central area had lower electron density than the circum area. The PDLSC-derived exosomes could express the common surface adhesion molecules CD9, CD63, CD81 and TSG101. NTA results showed that the collected exosomes had a size around (119±12.1) nm and an approximate concentration of (3.80±0.39)×10⁸ particles/mL. CONCLUSIONS: Exosomes derived from PDLSCs can be collected by a ultracentrifugation method, which expresses common membrane proteins and morphological characteristics of exosomes.

Key words: PDLSCs, Exosomes, Extracelluar vesicles, Ultracentrifugation

中图分类号:

R783.1

赵力如, 毛家奇, 赵丙姣, 陈静. 牙周膜干细胞来源外泌体的生物学特性分析[J]. 上海口腔医学, 2019, 28(4): 343-348.

ZHAO Li-ru, MAO Jia-qi, ZHAO Bing-jiao, CHEN Jing. Isolation and biological characteristics of exosomes derived from periodontal ligament stem cells[J]. Shanghai Journal of Stomatology, 2019, 28(4): 343-348.

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牙周膜干细胞来源外泌体的生物学特性分析

Isolation and biological characteristics of exosomes derived from periodontal ligament stem cells

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