中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (6): 1027-1030.doi: 10.3969/j.issn.1673-8225.2010.06.016

• 干细胞培养与分化 • 上一篇    下一篇

四种胞外基质支持人胚胎干细胞的生长：效果有差异吗？

胡智兴1，2，罗 敏2，3，周轶平2，3，梁道明4   

  1. 1昆明医学院药理学教研室，云南省昆明市  650031；
    2昆明医学院云南省生物医学工程中心，云南省昆明市650031；
    3昆明医学院云南省天然药物药理重点实验室，云南省昆明市  650031；
    4昆明医学院第二附属医院，云南省昆明市 650031
  • 出版日期:2010-02-05 发布日期:2010-02-05
  • 通讯作者: 通讯作者：梁道明，博士，副主任医师，昆明医学院第二附属医院，云南省昆明市 650031 liangdaoming@yahoo.com.cn
  • 作者简介:胡智兴，男，1970年生，浙江省永康市人，汉族，2007年中国科学院昆明动物研究所毕业，博士，讲师，主要从事干细胞方面的研究。 xingzhihu@163.com
  • 基金资助:

    云南省科技厅-昆明医学院应用基础研究联合专项基金资助项目(2007C0012R，2007C0044R)；
    云南省自然科学基金(2005C0013R)。

Four kinds of extracellular matrixes support the growth of human embryonic stem cells: Effect differences

Hu Zhi-xing1,2, Luo Min2,3, Zhou Yi-ping2,3, Liang Dao-ming4   

  1. 1Department of Pharmacology, Kunming Medical University, Kunming   650031, Yunnan Province, China;
    2Biomedical Engineering Research Center, Kunming Medical University, Kunming   650031, Yunnan Province, China; 
    3Yunnan Provincial Pharmacological Key Laboratory of Natural Drug, Kunming Medical University, Kunming   650031, Yunnan Province, China;
    4Second Affiliated Hospital, Kunming Medical University, Kunming 650031, Yunnan Province, China
  • Online:2010-02-05 Published:2010-02-05
  • Contact: Liang Dao-ming, Doctor, Associate chief physician, Second Affiliated Hospital, Kunming Medical University, Kunming 650031, Yunnan Province, China liangdaoming@yahoo.com.cn
  • About author:Hu Zhi-xing, Doctor, Lecturer, Department of Pharmacology, Kunming Medical University, Kunming 650031, Yunnan Province, China; Biomedical Engineering Research Center, Kunming Medical University, Kunming 650031, Yunnan Province, China xingzhihu@163.com
  • Supported by:

    Special Research Foundation of Department of Science and Technology of Yunnan Province-Kunming Medical University, No.2007C0012R, 2007C0044R**;
    Natural Science Foundation of Yunnan Province, No.2005C0013R*

PDF

498

摘要：

背景：胞外基质在维持人胚胎干细胞的自我更新和未分化状态方面具有重要作用，建立无血清无饲养层、成分明确的人胚胎干细胞培养系统是人胚胎干细胞运用于临床移植研究的前提。

目的：比较4种胞外基质对于维持人胚胎干细胞生长的效果差异。

方法：常规复苏人胚胎干细胞株BG02，设立4组，分别将人胚胎干细胞转至预铺有纤粘连蛋白、Ⅳ型胶原、Matrigel、层粘连蛋白的培养板上，均在含bFGF，TGFβ及ITS无血清培养基中培养。测定细胞集落贴壁率及分化率，流式细胞仪检测人胚胎干细胞特异性分子标志Oct-4及Nanog的表达，RT-PCR检测胞外基质受体的表达。

结果与结论：与纤粘连蛋白组、Ⅳ型胶原组比较，Matrigel组和层粘连蛋白组人胚胎干细胞集落的贴壁率均明显增加(P < 0.05)，分化率均明显降低(P < 0.05)，Oct-4，Nanog阳性率均明显升高(P < 0.05)，后2组各项指标比较无明显差异(P > 0.05)。Matrigel组、层粘连蛋白组人胚胎干细胞高表达整合素受体integrinα5，integrinα6和integrinβ1；而纤粘连蛋白组、Ⅳ型胶原组3种整合素受体的表达均明显降低。提示Matrigel和层粘连蛋白可以较好支持人胚胎干细胞在无饲养层体系生长，而纤粘连蛋白和Ⅳ型胶原无法维持人胚胎干细胞的未分化状态；人胚胎干细胞integrin受体家族激活状态的不同，可能是不同胞外基质对于维持人胚胎干细胞自我更新产生差异的原因之一。

关键词: 胞外基质, 自我更新, 生长, 整合素, 人胚胎干细胞

Abstract:

BACKGROUND: Extracellular matrixes play important roles in the maintenance of undifferentiated state and self-renewal of human embryonic stem cells (hESCs). Establishment of a defined serum- and feeder-free culture system is a prerequisite for the application of hESCs in cell-replacement therapy.

OBJECTIVE: The present study was designed to compare the effects of four kinds of extracellular matrixes on the support the growth of hESCs.

METHODS: We set four groups using conventionally recovered hESCs BGO2. BG02 was cultured on Matrigel-, laminin-, fibronectin-, or collagen-coated plates in serum-free medium containing basic fibroblast growth factor (bFGF), transforming growth factor beta 1(TGFβ1), Insulin-Transferrin-Selenium (ITS). The attachment rate and differentiated rate of BG02 clumps in various conditions were measured. The proportions of Oct-4 and Nanog positive cells in various groups were assessed by flow cytometry. The extracellular matrix receptor expression was analyzed by RT-PCR.

RESULTS AND CONCLUSION: The attachment rates of BG02 cell clumps in Matrigel and laminin groups were significantly higher than that of fibronectin and collagen groups (P < 0.05), but differentiated rate was significantly reduced (P < 0.05). The percentage of Oct-4+ and Nanog+ BG02 cells in Matrigel and laminin groups were higher than that of fibronectin and collagen groups (P < 0.05). No significant difference in each index was determined between the fibronectin and collagen groups (P > 0.05). BG02 cells in Matrigel and laminin groups expressed higher levels of integrin α5, integrin α6 and integrin β1 mRNA than that in fibronectin and collagen groups. Above-mentioned results indicated that Matrigel and laminin could support the proliferation of BG02 cells well, while fibronectin and collagen could not support the proliferation of BG02 cells. These variations might be attributed to the activation of integrin subunits in BG02 cells.

中图分类号: