中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (23): 4299-4302.doi: 10.3969/j.issn.1673-8225.2010.23.027

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

ICR小鼠胚胎成纤维细胞的分离培养及饲养层制备

方驰华1，胡海北1，郝建志1，陈 霞2   

  1. 南方医科大学附属珠江医院，1肝胆一科，2超声诊断科，广东省广州市  510180
  • 出版日期:2010-06-04 发布日期:2010-06-04
  • 通讯作者: 陈 霞，无，主管技师，南方医科大学附属珠江医院超声诊断科 superchenxia@ 163.com
  • 作者简介:方驰华，男，1958年生，湖北省汉川市人，汉族，2005年解放军第一军医大学毕业，博士，主任医师，教授，博士生导师，主要从事肝卵圆干细胞，骨髓间充质干细胞及数字医学的研究。 fangch?dr@126. com
  • 基金资助:

    广东省自然科学基金资助项目(8121051501000000)

Isolation and culture of ICR mouse embryonic fibroblasts and production of feeder layers

Fang Chi-hua1, Hu Hai-bei1, Hao Jian-zhi1, Chen Xia2   

  1. 1First Department of Hepatobiliary Surgery, 2Department of Ultrasound Diagnosis, Zhujiang Hospital Affiliated to Southern Medical University, Guangzhou 510180, Guangdong Province, China
  • Online:2010-06-04 Published:2010-06-04
  • Contact: Chen Xia, Technician-in-charge, Department of Ultrasound Diagnosis, Zhujiang Hospital Affiliated to Southern Medical University, Guangzhou 510180, Guangdong Province, China superchenxia@163. com
  • About author:Fang Chi-hua, Chief physician, Professor, Doctoral supervisor, First Department of Hepatobiliary Surgery, Zhujiang Hospital Affiliated to Southern Medical University, Guangzhou 510180, Guangdong Province, China fangch?_dr@126.com
  • Supported by:

     the Natural Science Foundation of Guangdong Province, No. 8121051501000000*

PDF

782

被引次数

20

摘要：

背景：小鼠胚胎成纤维细胞作为饲养层能有效促进胚胎干细胞及诱导多能性干细胞增殖并维持其未分化特性和多潜能性，且效果优于无饲养层单独添加一些细胞因子，而且可以为上述两种细胞提供类似的胚胎发育环境。

目的：建立稳定高效的ICR小鼠胚胎成纤维细胞饲养层培养体系，用于培养人胚胎干细胞及诱导多能性干细胞。

方法：使用胰蛋白酶消化法分离ICR小鼠胚胎成纤维细胞，观察不同的胰蛋白酶水平及消化时间对获取小鼠胚胎成纤维细胞的量及其增殖活性的影响；利用不同质量浓度丝裂霉素C处理小鼠胚胎成纤维细胞1，1.5，2，2.5，3，3.5 h制备饲养层细胞，MTT法检测其增殖活性，探索其制备饲养层的最佳条件。

结果与结论：分离ICR小鼠胚胎原代成纤维细胞最佳胰蛋白酶浓度为0.05%，消化时间为12~15 min，丝裂酶素最佳作用质量浓度和时间为10 mg/L作用2.5 h，成纤维细胞饲养层可以维持8~12 d。0.05%胰蛋白酶消化15~20 min效果优于0.15%，0.25%胰蛋白酶消化；10 mg/L丝裂霉素C处理胚胎成纤维细胞2.5 h能有效抑制其增殖，表明该条件下的饲养层细胞能很好的支持胚胎干细胞及诱导多能性干细胞生长。

关键词: 胚胎成纤维细胞, 丝裂霉素C, 饲养层, 胚胎干细胞, 诱导多能性干细胞

Abstract:

BACKGROUND: Mouse embryonic fibroblast (MEF) as feeder layer can be effective to promote embryonic stem cells and to induce pluripotent stem cell proliferation and to maintain their undifferentiated and pluripotent characteristics, which are better than feeder-free or adding single cytokines in medium, furthermore, the feeder layer can provide embryonic development environment for the above two kinds of cells.
OBJECTIVE: To establish a stable and effective system of ICR MEF feeder layers to support embryonic stem cells and induce pluripotent stem cells in vitro.
METHODS: MEF of ICR mouse was isolated using the method of trypsin digestion to observe effects of different mass concentrations of trypsin and different digestion time on the amount and the proliferation activity of MEF. Various concentrations of mitomycin C were utilized to treat MEF for 1, 1.5, 2, 2.5, 3, 3.5 hours to prepare feeder layer cells. MTT assay was employed to determine proliferation activity of MEF, and to explore optimal condition for preparing feeder layer.
RESULTS AND CONCLUSION: The optimal mass concentration of trypsin for isolating MEF of ICR mouse was 0.05%; digestion time was 12-15 minutes; the optimal action mass concentration and time was 10 mg/L for 2.5 hours. Fibroblast feeder layer maintained for 8-12 days. The effects of 0.05% trypsin digestion for 15-20 minutes were superior to high mass concentration.  10 mg/L mitomycin C treated MEF for 2.5 hours can effectively inhibit the proliferation of the prepared feeder cells. Feeder layer cells prepared under this condition can support embryonic stem cells and induce pluripotent stem cells growth

中图分类号: