上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2019, Vol. 39 ›› Issue (2): 126-.doi: 10.3969/j.issn.1674-8115.2019.02.004

• 论著·基础研究 • 上一篇    下一篇

人根尖乳头干细胞来源外泌体的提取及鉴定

晋巧巧，林文珍，苑克勇，牛晨光，黄正蔚   

  1. 上海交通大学附属第六人民医院妇产科，上海 200233
  • 出版日期:2019-02-28 发布日期:2019-03-19
  • 通讯作者: 黄正蔚，电子信箱：huangzhengwei@shsmu.edu.cn。
  • 作者简介:晋巧巧（1993—），女，硕士生；电子信箱： ersan61243578@sjtu.edu.cn。
  • 基金资助:
    国家自然科学基金（ 81570964，81371143）；上海市科学技术委员会基金（ 16430723500）

Extraction and identification of human stem cells the apical papilla derived-exosomes

JIN Qiao-qiao, LIN Wen-zhen, YUAN Ke-yong, NIU Chen-guang, HUANG Zheng-wei   

  1. Department of Endodontics, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, Shanghai 200011, China
  • Online:2019-02-28 Published:2019-03-19
  • Supported by:
    National Natural Science Foundation of China, 81570964, 81371143; Shanghai Municipal Commission of Science and Technology Foundation, 16430723500

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摘要： 目的 ·从人根尖乳头干细胞（human stem cells the apical papilla，hSCAPs）中提取外泌体并进行鉴定。方法 ·使用改良组织块法培养 hSCAPs并检测干细胞表面标志物 CD105、CD45、CD44、CD31、CD34、CD29的表达情况。体外诱导 hSCAPs向成骨细胞和脂肪细胞分化以检测其多向分化能力。使用梯度离心方法从 hSCAPs培养上清中分离出外泌体。通过粒径分析确定外泌体粒径大小，透射电镜观察外泌体形态特征， Western blotting进行外泌体特异性抗原分子 CD81、CD9、CD63和 TSG101的鉴定。结果 ·在 hSCAPs中检测到间充质干细胞表面抗原标志物 CD105、CD44和 CD29表达呈强阳性，而造血干细胞标志物 CD45、CD31和 CD34呈阴性。 hSCAPs能够向成骨细胞和脂肪细胞分化。从 hSCAPs的上清中分离出来的囊泡样物质，其外形为圆形囊泡并且具有完整的膜结构，粒径为 30～ 100 nm，而且表达外泌体特性蛋白 CD81、CD9、CD63和 TSG101。结论 ·成功地从 hSCAPs中分离出囊泡样物质，并鉴定其为外泌体。

关键词: 人根尖乳头干细胞, 外泌体, 梯度离心, 鉴定

Abstract:

Objective · To obtain and identify the exosomes derived human stem cells the apical papilla (hSCAPs). Methods · hSCAPs were culturedmodified tissue adherence method and the phenotypes were analyzed with stem cell surface markers CD105, CD45, CD44, CD31, CD34 and CD29. The capability of multi-differentiation in hSCAPs was identifiedosteogenic and adipogenic differentiation in vitro. Exosomes were isolated hSCAPs culture supernatants using gradient centrifugation methods. The size of vesicle was assessednanoparticle size analyzer. The morphology of exosomes was observedtransmission electronic microscope (TEM), and the of exosome molecular markers CD81, CD9, CD63 and TSG101 was analyzedWestern blotting. Results · hSCAPs were positive for the mesenchyme stem cell markers, including CD105, CD44 and CD29 and negative for the hematopoietic markers CD45, CD31 and CD34. hSCAPs could differentiate into osteoblasts and adipocytes. hSCAPs secreted microvesicles which exhibited round vesicle structure with an intact membrane observedthe TEM. The results of nanoparticle size analyzer measurement showed that the diameters of vesicles were ranged 30 to 100 nm, which were consistent with the resultsTEM. Microvesicles could express the molecular markers for exosomes, i.e. CD81, CD9, CD63 and TSG101. Conclusion · The microvesicles were successfully isolated hSCAPs and identified as exosomes.

Key words: human stem cell the apical papilla, exosome, gradient centrifugation, identification

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