中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (45): 8429-8433.doi: 10.3969/j.issn.1673-8225.2011.45.015

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

鸡胚胎干细胞分离方法和培养体系的优化

张曼玉1，陈志胜2，计慧琴2，陈胜峰2，刘本杰1，邓衔柏1，王丙云2，袁  立3，江青艳4   

  1. 1华南农业大学兽医学院，广东省广州市   510642
    2佛山科学技术学院生命科学学院动物医学系，广东省佛山市   528231
    3厦门大学生命科学学院生物医学系，福建省厦门市   361005
    4华南农业大学动物科学学院，广东省广州市  510642
  • 收稿日期:2011-05-06 修回日期:2011-06-18 出版日期:2011-11-05 发布日期:2011-11-05
  • 通讯作者: 邓衔柏，硕士，副教授，硕士生导师，华南农业大学兽医学院，广东省广州市 510642 xbdeng@scau.edu.cn 并列通讯作者：王丙云，博士，教授，硕士生导师，佛山科学技术学院生命科学学院动物医学系，广东省佛山市 528231 bywang63@163.com
  • 作者简介:张曼玉★，女，1986年生，山东省临沂市人，汉族，华南农业大学兽医学院在读硕士，主要从事干细胞研究。 linyizhangmanyu@163.com
  • 基金资助:

    国家973项目(2009CB941600)资助；国家自然科学基金项目(31072101)资助。

Optimization of separation methods and culture system of chicken embryonic stem cells in vitro

Zhang Man-yu1, Chen Zhi-cheng2, Ji Hui-qin2, Chen Sheng-feng2, Liu Ben-jie1, Deng Xian-bo1, Wang Bing-yun2, Yuan Li3, Jiang Qing-yan4   

  1. 1Veterinary College, South China Agricultural University, Guangzhou  510642, Guangdong Province, China
    2Veterinary Department, College of Life Science, Foshan University, Foshan  528231, Guangdong Province, China
    3Department of Biomedicine, School of Life Science, Xiamen University, Xiamen  361005, Fujian Province, China
    4College of Animal Science, South China Agricultural University, Guangzhou  510642, Guangdong Province, China
  • Received:2011-05-06 Revised:2011-06-18 Online:2011-11-05 Published:2011-11-05
  • Contact: Deng Xian-bo, Master, Associate professor, Master’s supervisor, Veterinary College, South China Agricultural University, Guangzhou 510642, Guangdong Province, China xbdeng@scau.edu.cn Correspondence to: Wang Bing-yun, Doctor, Professor, Master’s supervisor, Veterinary Department, College of Life Science, Foshan University, Foshan 528231, Guangdong Province, China bywang63@163.com
  • About author:Zhang Man-yu★, Studying for master’s degree, Veterinary College, South China Agricultural University, Guangzhou 510642, Guangdong Province, China Linyizhangmanyu @163.com
  • Supported by:

    the National 973 Program of China, No. 2009CB941600*; the National Natural Science Foundation of China, No. 31072101*

PDF

686

被引次数

18

摘要：

背景：胚胎干细胞是从动物早期胚胎的内细胞团或原始生殖细胞分离出来的具有发育全能性的一种未分化的无限增殖细胞系。而鸡胚胎干细胞则是从X期鸡胚的胚盘分离而来。
目的：优化鸡胚胎干细胞分离方法和离体培养体系。
方法：采用滤纸纸环-发环的方法从X期鸡胚分离胚盘细胞,并采用STO细胞作为饲养层和大鼠肝细胞(BRL)条件培养基(CM)+细胞因子作为离体培养体系对分离的胚盘细胞进行培养。
结果与结论：滤纸纸环-发环法获得的完整胚盘率为75%~85%，克隆形成率约为50%。BRL-CM+饲养层培养体系，鸡胚胎干细胞可传至7代，而BRL-CM+饲养层+细胞因子培养体系，鸡胚胎干细胞可传至25代。分离到的鸡胚胎干细胞，经碱性磷酸酶染色、SSEA-1染色鉴定，表明鸡胚胎干细胞处于未分化状态。提示，实验不仅优化了鸡胚胎的分离方法，获得完整且杂质少的胚盘，而且进一步优化了鸡胚胎干细胞体外培养体系。

关键词: 鸡, 胚胎干细胞, 分离, 培养, 细胞因子

Abstract:

BACKGROUND: Embryonic stem cells are undifferentiated permanent cell line derived from inner cell mass cells and primordial germ cells of animal’s early embryos. Chicken embryonic stem cells are derived from the blastodermal of a X-stage embryo.
OBJECTIVE: To optim the separation method and in vitro cultural system of chicken embryonic stem cells.
METHODS: The X-stage chicken embryos were isolated by using a small square of ?lter paper with a hole punched in the center, and the blastodermal cells were isolated by using the hair loop. STO cells were used to make feeder layer; at the same time, BRL-CM and cytokine were also used for chicken embryonic stem cells in vitro cultural system.
RESULTS AND CONCLUSION: The filter paper loop and the hair loop could obtain complete the blastoderm, and the successful percentage was 75%-85%. The colony formation rate was about 50%. After culture in the BRL-CM + feeder layer + cytokine culture system, the passage of CES cells is the seventh generation; BRL-CM + feeder layer + cytokines, cultured chicken embryonic stem cells could passage to the 25th generation. Isolated chicken embryonic stem cells were in an undifferentiated state detected by alkaline phosphatase staining and SSEA-1 staining. The findings indicate that this experiment not only optimized the isolation method of chicken embryonic stem cells to obtain complete and pure embryos, but also further improved the in vitro culture system of chicken embryonic stem cells.

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