中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (23): 4303-4308.doi: 10.3969/j.issn.1673-8225.2010.23.028

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

小鼠饲养层细胞制备与小鼠胚胎干细胞SF1-G的培养

刘  峰，雷闽湘，陈慧玲   

  1. 中南大学湘雅医院内分泌科，湖南省长沙市 410008
  • 出版日期:2010-06-04 发布日期:2010-06-04
  • 通讯作者: 陈慧玲，博士，副主任医师，中南大学湘雅医院内分泌科，湖南省长沙市 410008 huilingcheng_8@hotmail.com
  • 作者简介:刘 峰，男，1980年生，江西省安福县人，汉族，中南大学在读博士，主要从事糖尿病及其并发症的研究。 liufengdyx@163.com
  • 基金资助:

    国家自然科学基金课题(30771025)

Preparation of mouse feeder layer cells and culture of mouse embryonic stem cell SF1-G

Liu Feng, Lei Min-xiang, Chen Hui-ling   

  1. Department of Endocrinology, Xiangya Hospital, Central South University, Changsha  410008, Hunan Province, China
  • Online:2010-06-04 Published:2010-06-04
  • Contact: Chen Hui-ling, Doctor, Associate chief physician, Department of Endocrinology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China huilingcheng_8@ hotmail.com
  • About author:Liu Feng, Studying for doctorate, Department of Endocrinology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China liufengdyx@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30771025*

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被引次数

2

摘要：

背景：小鼠胚胎干细胞系SF1-G是由雌性C57BL/6 小鼠与雄性M. spretus 小鼠交配后，取桑葚胚期胚胎在STO饲养层细胞上分离培养获得，STO细胞较昂贵，而由小鼠胚胎成纤维细胞制备的饲养层细胞不仅取材容易，而且形成胚胎干胞的克隆率、维持胚胎干细胞正常核型的能力均比STO细胞要好一些，因此，建立一种适宜SF1-G细胞扩增的培养体系，保持其未分化状态生长是充分利用胚胎干细胞资源的前提。
目的：建立有效的小鼠胚胎成纤维细胞的分离培养及胚胎干细胞饲养层细胞制备体系；以建立有效的小鼠胚胎干细胞(SF1-G细胞)扩增培养体系。
方法：从孕12.5~14.5 d的ICR小鼠分离培养原代小鼠胚胎成纤维细胞；取 3~5 代的小鼠胚胎成纤维细胞，以丝裂霉素C抑制其增殖能力制备饲养层细胞；在饲养层细胞上增殖培养SF1-G细胞；染色体G显带分析法检测SF1-G细胞核型，SF1-G细胞碱性磷酸酶染色和RT-PCR检测Oct4、Nanog基因表达。
结果与结论：从孕鼠胚胎有效分离到小鼠胚胎成纤维细胞，以3~5代细胞制备的饲养层细胞能够支持胚胎干细胞SF1-G呈边界清晰的克隆样生长。染色体核型检测 SF1-G保持正常核型，碱性磷酸酶、表面标志物检测均呈阳性。实验建立了有效的小鼠胚胎成纤维细胞分离培养体系，并制备供胚胎干细胞进行增殖培养饲养层细胞体系，能够在实验室对 SF1-G细胞保持正常未分化状态培养。

关键词: 小鼠, 胚胎成纤维细胞, 饲养细胞, 胚胎干细胞, 细胞培养

Abstract:

BACKGROUND: Mouse embryonic stem cell line SF1-G is derived from morula of female C57BL/6 mice mating with male M. spretus. Usually SF1-G was cultured on the layer of STO feeders. STO cells were expensive, whereas mouse embryonic fibroblasts (MEFs) were easier to get, and had more advantage in supporting embryonic stem cells growth, such as the formation of embryonic stem cell clones and maintaining the normal karyoplast of embryonic stem cells. Thus, it was very useful to establish a culture system in which SF1-G cells could amplify in an undifferentiated status.

OBJECTIVE: To explore and establish an effective method of isolating and culturing MEFs and prepare the feeder cells for mouse embryonic stem cell (SF1-G cells) proliferation. 

METHODS: The mouse primary MEFs were isolated from ICR mouse fetus at 12.5 to 14.5 day gestational ages. The 3 to 5 passages MEFs were treated by mytomycin C to inhibit cell proliferation. The treated MEFs were used as feeder cells for culturing SF1-G cell. The karyotype of SF1-G cell was detected by chromosome G staining process. The expression of alkaline phosphatase (AKP) of SF1-G cells was tested. Oct4 and Nanog gene expressions were also tested by RT-PCR.

RESULTS AND CONCLUSION: MEFs were successfully isolated and cultured from mouse fetus. Feeder cells prepared MEFs of 3 to 5 passages could be used to support SF1-G cell growth with clear boundaries of clone. The SF1-G cells had normal karyotype, showing the positive results of AKP and expressing specific transcription factors. This experiment established an effective method of isolating and culturing MEFs and prepared feeder cells for propagating and culturing mouse embryonic stem cells, and SF1-G cells can be cultured in undifferentiated state in a laboratory.

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