基础医学与临床 ›› 2008, Vol. 28 ›› Issue (3): 264-268.

• 技术与方法 • 上一篇    下一篇

体外构建胚胎干细胞生长分化的三维研究模型

魏国峰 李向东 张维疆 曲鹏   

  1. 大连医科大学附属二院 大连医科大学附属二院 大连医科大学附属二院 大连医科大学附属二院
  • 收稿日期:2006-12-27 修回日期:2007-06-15 出版日期:2008-03-25 发布日期:2008-03-25
  • 通讯作者: 曲鹏

Construction a Three Dimensional Culture Model for Inducing Embryonic Stem Cells Differentiation in vitro

Guo-feng WEI, Xiang-dong LI, Wei-jiang ZHANG, Peng QU   

  1. Dept. of Cardiology, the Second Affiliated Hospital of Dalian Medical University
  • Received:2006-12-27 Revised:2007-06-15 Online:2008-03-25 Published:2008-03-25
  • Contact: Peng QU

摘要: 目的 以液态胶原为支架,小鼠胚胎干细胞(ESCs)为细胞模型,构建ESCs-胶原复合体,旨在尝试建立一种能够实现干细胞生长、分化的三维培养体系。方法 提取鼠尾胶原,观察ESC在自制胶原条带内增殖的形态特征,并测定葡萄糖/乳酸活性。以ESCs源心肌细胞为目的细胞,利用免疫组化、RT-PCR及电镜技术评价胶原条带内ESCs进行自发性分化的能力。结果 ESCs在胶原条带所提供的三维培养体系内生长、增殖状态良好,且彼此间能够建立细胞连接。胶原条带内部分ESCs能够自发性分化为心肌细胞。该心肌细胞均可表达心肌蛋白cTn-T,心肌转录因子NKX2.5 及肌球蛋白轻链MLC-2v mRNA,且肌小节结构发育成熟。结论 以液态I型胶原为主体的支架材料可以为ESCs提供良好的生长基质,促进其组织化发育。该实验初步明确了体外构建干细胞生长分化三维模型的可行性。

关键词: 胚胎干细胞, 胶原, 三维培养, 分化

Abstract: Objective To mimic the three-dimensional (3-D) growth pattern of stem cells in vivo, a 3-D culture system in vitro constructed by rat tail collagen scaffold was established. Methods Circular strands were prepared by mixing suspended mouse embryonic stem cells (mESCs) with rat tail collagen. Growth profile of mESCs within the collagen strand was observed under phase contrast microscope. Their metabolic activity was evaluated by glucose/lactic acid contents. To evaluate the effect of 3D culture system on ESCs differentiation, ES-derived cardiomyocytes were detected by immunohistochemistry, RT-PCR and transmission electron microscope (TEM), respectively. Results ESCs grew well within the 3-D culture system constructed by rat tail collagen. As time passage, cell connections can be found in those cell clusters formed within collagen stands, which indicated that tissue-like cultures should be produced during the process of 3-D culture in vitro. Further, ESCs cultured by 3-D collagen strand differentiated into cardiomyocytes spontaneously. Conclusions Collagen could provide an ideal growth matrix for ESCs proliferation in vitro and promote ESCs differentiation towards tissue-like structures. Thus, three dimensional culture system constructed by rat tail collagen could be applied to study ESCs differentiation mimicking in vivo.

Key words: embryonic stem cells (ESC), collagen, three dimensional culture, differentiation