中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (1): 49-53.doi: 10.12307/2022.983

• 牙髓及牙周膜干细胞 Dental pulp and periodontal ligament stem cells • 上一篇    下一篇

不同来源条件培养基对人牙髓干细胞增殖的影响

杨  燕1,王静娴1,张荣红1,李  晨2,范安然1,崔冬冰1,吴淑梅3   

  1. 1贵州医科大学,贵州省贵阳市   550004;2长江航运总医院,湖北省武汉市   430014;3贵州省第二人民医院,贵州省贵阳市   550004
  • 收稿日期:2021-12-17 接受日期:2022-01-30 出版日期:2023-01-08 发布日期:2022-06-06
  • 通讯作者: 吴淑梅,副主任医师,贵州省第二人民医院,贵州省贵阳市 550004
  • 作者简介:杨燕,女,1980年生,贵州省黄平县人,汉族,2021年贵州医科大学毕业,实验师,主要从事干细胞研究。
  • 基金资助:
    贵州省教育厅青年科技人才成长项目(黔教合KY字[2017]167),项目负责人:杨燕

Effects of conditioned media of different sources on the proliferation of human dental pulp stem cells

Yang Yan1, Wang Jingxian1, Zhang Ronghong1, Li Chen2, Fan Anran1, Cui Dongbing1, Wu Shumei3   

  1. 1Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 2General Hospital of The Yangtze River Shipping, Wuhan 430014, Hubei Province, China; 3Second People’s Hospital of Guizhou Province, Guiyang 550004, Guizhou Province, China
  • Received:2021-12-17 Accepted:2022-01-30 Online:2023-01-08 Published:2022-06-06
  • Contact: Wu Shumei, Associate chief physician, Second People’s Hospital of Guizhou Province, Guiyang 550004, Guizhou Province, China
  • About author:Yang Yan, Experimentalist, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Supported by:
    the Youth Science and Technology Talents Growth Project of Guizhou Provincial Department of Education, No. KY[2017]167 (to YY)

摘要:

文题释义:
牙髓干细胞:是来源于牙髓组织的成体干细胞,该细胞与骨髓间充质干细胞有着相似的免疫表型、细胞形态,具有自我更新、多向分化潜能,以及较强的克隆、再生修复能力。
实时无标记细胞分析仪(RTCA):是一种检测贴壁活细胞生长状态的新技术,它是建立在阻抗基础上的瞬时细胞电感应连续记录系统,电阻与细胞是否黏附、数量、体积大小及形态变化密切相关,通过检测电阻值进而实时动态监测细胞生长情况,具有实时、无标记、无损伤的特点。

背景:利用条件培养基与人牙髓干细胞共培养,初步探索可以快速促进人牙髓干细胞增殖的方法,为今后细胞治疗、扩增高质量种子细胞提供研究基础。
目的:初步探究不同来源条件培养基对人牙髓干细胞增殖的影响。
方法:体外分离培养人牙髓干细胞,并用流式细胞术鉴定。然后将胎牛血清超高速低温离心产物、骨折患者尿液超高速低温离心产物、骨髓来源树突状细胞体外培养上清液、人皮肤成纤维细胞培养上清液与低糖DMEM培养液配置成条件培养基后,再分别与人牙髓干细胞共培养,使用细胞无标记培养观察装置(BioStation-T)连续拍摄各组细胞生长72 h的照片、使用实时无标记细胞分析仪(RTCA)动态监测150 h,比较各组细胞的标准化细胞指数值的差异。
结果与结论:①人牙髓干细胞为典型的间充质干细胞形态,表达间充质干细胞表面标志物CD90和CD105,不表达CD34和CD45;②细胞无标记培养观察装置动态监测时,发现胎牛血清超高速低温离心组的颗粒物质被吸收时间要显著早于其他实验组和空白对照组;③在实时无标记细胞分析仪检测时,与空白对照组相比,胎牛血清超高速低温离心组、骨髓来源树突状细胞体外培养上清液组、人皮肤成纤维细胞培养上清液组的标准化细胞指数值均显著升高(P < 0.001),骨折患者尿液超高速低温离心组的标准化细胞指数值显著降低(P < 0.001);④结果表明,胎牛血清超高速低温离心产物、骨髓来源树突状细胞体外培养上清液、人皮肤成纤维细胞培养上清液所配置的条件培养基能促进人牙髓干细胞增殖,其中胎牛血清超高速低温离心产物所配置的条件培养基对细胞还有一定保护作用。

https://orcid.org/0000-0002-1487-8805 (杨燕)

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

关键词: 人牙髓干细胞, 超高速低温离心, 条件培养基, 细胞增殖, 标准化细胞指数, 组织块培养法

Abstract: BACKGROUND: Using conditioned medium and human pulp stem cells, we will initially explore methods to rapidly proliferate human dental pulp stem cells to provide a research basis for future cell treatment and expansion of high-quality seed cells.  
OBJECTIVE: To preliminarily explore the effect of conditioned medium from different sources on the proliferation of human dental pulp stem cells.   
METHODS: Human dental pulp stem cells were isolated in vitro and identified by flow cytometry. Fetal bovine serum ultra-high-speed cryogenic centrifugal products, urine ultra-high-speed cryogenic centrifugal products of fracture patients, in vitro culture supernatants of bone marrow-derived dendritic cells, human skin fibroblast cultures and low-glucose DMEM medium were configured into conditioned medium. After co-culture with human pulp stem cell cells, images of cell growth in each group were taken continuously for 72 hours using a cell label-free culture observation device (BioStation-T). The cells were dynamically monitored using a real-time label-free cell analyzer for 150 hours. Differences in normalized cell index values were compared between each group of cells.
RESULTS AND CONCLUSION: (1) Human dental pulp stem cells are typical of mesenchymal stem cells. Expression of the mesenchymal stem cell surface markers CD90 and CD105 was found, but CD34 and CD45 expression was not observed. (2) Cell label-free culture observation device when dynamically monitoring found that the absorption time in the group was significantly earlier than in other experimental and blank control groups. (3) When detected on a real-time label-free cell analyzer, compared with the blank control group, the normalized cell index values of the fetal bovine serum ultra-high-speed cryogenic centrifugal group, the bone marrow-derived dendritic cell in vitro culture supernatant group, and the human skin fibroblast culture supernatant group were significantly increased (P < 0.001). The normalized cell index value of fracture patients of the urine ultra-high-speed cryogenic centrifugal group was significantly decreased (P < 0.001). (4) The results show that the conditioned medium configured by ultra-high-speed centrifugation, in vitro culture supernatant of bone marrow-derived dendritic cells, and human skin fibroblasts can promote the proliferation of human dental pulp stem cells. Among them, the conditioned medium configured by the fetal bovine serum ultra-high-speed cryogenic centrifugal products also had some protective effects on the cells. 

Key words: human dental pulp stem cell, ultra-high-speed cryogenic centrifugation, conditioned medium, cell proliferation, normalized cell index, tissue culture method

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