上海交通大学学报（医学版）

›› 2009, Vol. 29 ›› Issue (7): 808-.

• 论著（基础研究） • 上一篇    下一篇

类胚体中残留未分化胚胎干细胞的初步研究

付 炜, 皮庆猛, 石伦刚, 唐郑雅, 曹谊林, 张文杰   

  1. 上海交通大学 医学院第九人民医院整复外科 组织工程国家工程研究中心, 上海 200011
  • 出版日期:2009-07-25 发布日期:2009-09-16
  • 通讯作者: 张文杰, 电子信箱：wenjieboshi@yahoo.com.cn。
  • 作者简介:付 炜（1982—）, 男, 博士生；电子信箱: fuweizhulu@163.com。
  • 基金资助:

    国家重点基础研究发展计划（“973”计划）（2005CB522705）；国家自然科学基金（30571925，30671051）；上海市曙光人才计划（06SG22）

Residual undifferentiated embryonic stem cells in embryoid bodies

FU Wei, PI Qing-meng, SHI Lun-gang, TANG Zheng-ya, CAO Yi-lin, ZHANG Wen-jie   

  1. Department of Plastic and Reconstructive Surgery, National Tissue Engineering Center,The Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200011
  • Online:2009-07-25 Published:2009-09-16
  • Supported by:

    National Basic Research Program of China, 2005CB522705; National Natural Science Foundation of China, 30571925, 30671051; Shanghai Shuguang Talent Plan, 06SG22

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摘要：

目的 探讨类胚体中残留未分化胚胎干细胞（ESC）的特性。方法 小鼠R1和Oct-4-GFP转基因ESC细胞株，体外类胚体分化20 d后消化打散，重新给予ESC常规培养液培养。观察类胚体中残留未分化细胞形态；流式细胞仪和免疫荧光染色检测和观察残留细胞表面标志物及体外再次分化能力。将残留细胞扩增后注射入裸鼠背部皮下和大腿肌肉内，6周后注射部位取材进行大体和组织学检查。结果 分化20 d的类胚体中存在残留未分化ESC，生长形态呈克隆样，表达SSEA1、CD31、CD9和Oct-4等未分化ESC标志；细胞能反复传代，并可在体外再次分化和残留。残留细胞注射部位形成畸胎瘤，瘤体组织中存在成熟的内胚层、中胚层和外胚层组织。结论 胚胎干细胞分化为类胚体后仍存在残留未分化全能ESC，残留细胞在体内、外可再次分化，并能在体外分化中再次残留。

关键词: 胚胎干细胞, 分化, 类胚体, 残留

Abstract:

Objective To explore the residual undifferentiated mouse embryonic stem cells (ESCs) in embryoid bodies. Methods Mouse R1 and Oct-4-GFP transgenic ESCs were firstly cultured in suspension to form embryoid bodies (EBs). Twenty days later, EBs were digested into single cells and then  re-plated in standard ESC culture condition. The morphology of residual undifferentiated cells in EBs was observed, and surface makers and in vitro redifferentiation potency of residual cells were examined by flow cytometry and immunofluorescent staining. The residual cells were expanded and subcutaneously injected into nude mice, and the specimens were  harvested from the injection site for histological analysis 6 weeks after injection. Results There were residual undifferentiated ESCs in EBs differentiated for 20 days, which displayed clonal morphology and expressed undifferentiated cell markers of ESCs, including SSEA1, CD31, CD9 and Oct-4. The cells could be differentiated to form EBs again, and could be re-expanded from secondary EBs. The residual cells were able to form teratoma at the injection site, and mature endoderm, mesoderm and ectoderm tissues could be found in teratoma tissues. Conclusion There are residual undifferentiated ESCs after differentiation of ESCs into EBs. The residual ESCs can differentiate again in vitro and in vivo, and can residue again in the in vitro differentiation.

Key words: embryonic stem cell, differentiation, embryoid body, residue