中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (9): 1432-1438.doi: 10.3969/j.issn.2095-4344.2017.09.022

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

EdU标记兔外周血单个核细胞的体外增殖

赵鸣雷，甄栋钦，黄健发，黎韦华，王文聪，李志权，张和宁，冼碧琨，彭宇婷，周敏仪，黄  冰   

  1. 中山大学中山眼科中心，眼科学国家重点实验室，广东省广州市  510060
  • 出版日期:2017-03-28 发布日期:2017-03-31
  • 通讯作者: 黄冰，研究员，中山大学中山眼科中心，眼科学国家重点实验室，广东省广州市 510060
  • 作者简介:赵鸣雷，男，1991年生，江苏省江阴市人，汉族，中山大学在读硕士，主要从事干细胞与组织工程相关研究。
  • 基金资助:

    广东省科技计划项目(2013B040200020，2013B020400003)；广州市科技计划项目(15570001)

In vitro EdU labeling of peripheral blood mononuclear cells in rabbits

Zhao Ming-lei, Zhen Dong-qin, Huang Jian-fa, Li Wei-hua, Wang Wen-cong, Li Zhi-quan, Zhang He-ning,Xian Bi-kun, Peng Yu-ting, Zhou Min-yi, Huang Bing   

  1. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China
  • Online:2017-03-28 Published:2017-03-31
  • Contact: Huang Bing, Researcher, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China
  • About author:Zhao Ming-lei, Studying for master’s degree, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China
  • Supported by:

    the Scientific Plan Project of Guangdong Province, No. 2013B040200020, 2013B020400003; the Scientific Plan Project of Guangzhou City, No. 15570001

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摘要：

文章快速阅读：

文题释义：
外周血单个核细胞：是外周血细胞中具有圆形核的细胞群，主要由淋巴细胞(T细胞，B细胞，NK细胞)、单核细胞及少部分干细胞所组成，其在自身免疫疾病、传染病、恶性肿瘤、疫苗开发、移植免疫及组织修复等研究邻域中是重要的研究对象，具有潜在临床应用价值。
EdU：是一种尿嘧啶类似物，在与细胞孵育的过程中，随着细胞增殖、DNA复制，能渗入新合成的DNA中，使细胞获得标记，所以能带上EdU的细胞也是增殖的细胞；其与BrdU是同一类物质，但检测方法要简单的多，不需要DNA变性又能保存细胞良好的结构；是一种有效的、快速检测细胞增殖的物质，也可作为细胞体内移植的一种示踪剂。

 

摘要
背景：外周血单个核细胞群中含有外周血干细胞，其体外增殖情况仍不清楚，体内移植也缺乏良好的标记物。
目的：以EdU作为标记物，观察外周血单个核细胞在干细胞培养基中的增殖情况，以及探讨EdU标记外周血干细胞的可行性。   
方法：分离新西兰兔外周血单个核细胞，在加入EdU的干细胞培养液中培养1-5 d；于不同的时间点观察细胞形态和进行细胞计数；收获细胞，进行EdU染色，观察EdU阳性细胞并检测阳性率。
结果与结论：①刚分离出的外周血单个核细胞为类圆形，轮廓清晰；经过1 d培养时，大部分细胞仍悬浮在培养液中，球形或类圆形，有细胞团出现，也见少量细胞散在贴壁，呈现三角形或多边形；培养2 d时，可见较多细胞碎片，大多数细胞仍为类圆形；培养3-5 d时，细胞碎片增多，细胞团体积变小，细胞密度明显降低；②随着培养时间的延长，细胞计数逐渐降低；③在培养1 d时，有散在EdU红色标记细胞；在培养2 d时，EdU红色标记细胞明显增多，并在培养三四天时维持不明显的变化；在培养5 d时，EdU红色标记的细胞明显减少；培养2 d时，EdU阳性细胞率最高，为(2.38±0.10)%；④结果表明，外周血单个核细胞群中增殖细胞率很低，EdU能标记外周血单个核细胞中有增殖能力的细胞。

 

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

ORCID: 0000-0003-4315-7529(黄冰)

关键词: 干细胞, 分化, 外周血单个核细胞, EdU标记, 成体干细胞, 细胞增殖, 细胞移植

Abstract:

BACKGROUND: The proliferation of peripheral blood stem cells among peripheral blood mononuclear cells (PBMCs) in vitro remains unclear. There is no optimal marker for tracing PBMCs transplanted in vivo.
OBJECTIVE: To observe the degree of PBMC proliferation in stem cell medium by EdU labeling and to explore the feasibility of EdU-labeled peripheral blood stem cells.
METHODS: New Zealand rabbit PBMCs were isolated and cultured for 1 to 5 days in stem cell medium supplemented with EdU. The cells were observed and counted at 0, 1, 2, 3, 4 and 5 days in culture. The cells were harvested at each time point and stained with EdU fluorescent reagents. Then, confocal microscopy and flow cytometry were used to detect EdU-labeled cells.
RESULTS AND CONCLUSION: (1) Freshly isolated rabbit PBMCs were rounded and showed clear outline. After 1 day culture, most of the cells were suspended in the medium, spherical or round. There were also a few cell clusters and adherent cells scattered in a triangle or polygon shape; after 2 days culture, more cell debris were observed, and most cells were round; when cultured for 3-5 days, increased cell debris, smaller cell mass and decreased cell density significantly were observed. (2) With the prolongation of culture time, the cell count decreased gradually. (3) When cultured for 1 day, EdU labeled cells in red were scattered. The number of cells marked with EdU red label increased significantly at day 2 and remained unchanged after 3 days of culture. At 5 days of culture, the number of red cells markedly decreased; the highest positive rate of EdU-labeled cells was (2.38±0.10)% at 2 days after culture. To conclude, these results showed that the proportion of proliferating cells in rabbit PBMCs was very low. EdU is capable of labeling proliferative cells among PBMCs.

 

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

Key words: Mononuclear cells, Cell proliferation, Tissue engineering

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