小鼠ES细胞条件培养基对绵羊类胚胎干细胞克隆、传代的影响

畜牧兽医学报 ›› 2009, Vol. 40 ›› Issue (9): 1327-1332.doi:

小鼠ES细胞条件培养基对绵羊类胚胎干细胞克隆、传代的影响

白昌明^1,3,王新庄¹*,闫颖颖²,和小娥¹,张静芳²

1. 河南农业大学牧医工程学院，郑州 450002；2. 西北农林科技大学动物医学院, 杨凌 712100；3. 中国科学院动物研究所，北京 100101

收稿日期:2008-09-05 修回日期:1900-01-01 出版日期:2009-09-24 发布日期:2009-09-24
通讯作者: 王新庄

The Effects of Mouse ES Cell Conditioned Medium on Isolation and Cloning of Sheep Embryonic Stemlike Cells

BAI Changming ^1,3，WANG Xinzhuang ¹*，YAN Yingying²，HE Xiaoe ¹，ZHANG Jingfang²

1. College of Husbandry and Animal Medicine Engineering，Henan Agricultural University， Zhengzhou 450002, China; 2. College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling 712100, China; 3. Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

Received:2008-09-05 Revised:1900-01-01 Online:2009-09-24 Published:2009-09-24
Contact: WANG Xinzhuang

摘要/Abstract

摘要：

本研究以无血清培养基为基础培养液，旨在探求小鼠ES细胞条件培养液（ESCCM）和2种不同饲养层对绵羊类ES细胞分离、克隆效率的影响。绵羊内细胞团从胚胎中分离得到后，分别以小鼠胎儿成纤维细胞（MEF）和绵羊胎儿成纤维细胞（SEF）为饲养层进行培养。结果在MEF饲养层上，绵羊类ES细胞在ESCCM新培养系统中可稳定传至第10代，而使用基础培养液最多传至5代。而在SEF饲养层上，绵羊类ES细胞在ESCCM新培养系统中仅能传至3代。表明使用ESCCM和MEF能促进绵羊类ES细胞的分离和克隆。对类ES细胞进行核型分析、AKP染色及体外分化能力检测，证实所分离的类ES细胞符合ES细胞的主要特征，而且发现这些类ES细胞可以表达胚胎干细胞关键转录因子Nanog。结果表明，ESCCM可显著提高绵羊类ES细胞的分离克隆效率，原因可能是小鼠ES细胞在生长过程中可能分泌某些重要的细胞因子，从而达到促进绵羊ES细胞增殖的作用。且MEF比SEF更适合于绵羊类ES细胞的分离和传代。

Abstract:

In order to evaluate the efficiency of mouse embryonic stem cells conditioned medium (ESCCM) and two kinds of feeder layers on selfrenewal of sheep embryonic stemlike cells, serumfree media was used to derive sheep embryonic stem (ES)like cells from embryos. Inner cell masses (ICM) were isolated and grown on the mouse embryonic fibroblasts (MEF) and sheep embryonic fibroblasts (SEF) as feeder layers respectively. The sheep ESlike cells were found to be easily obtained and remained undifferentiated for 10 passages in the new culture system with MEF, and 3 passages with SEF feeder layer. However, when cultured in medium without ESCCM, goat ESlike cells could not survive for more than 5 passages. Sheep ESlike cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ESlike cells was confirmed by expressing a novel ES cells specifically expressed transcription factor (Nanog) and forming embryoid bodies in suspension culture. These results suggest that mouse ES cells might secrete some factors which play important roles in promoting sheep ESlike cells′ selfrenewal. Additionally, the results also indicated that MEF is more suitable for the isolation and passage of sheep ESlike cells.

白昌明;王新庄;闫颖颖;和小娥;张静芳 . 小鼠ES细胞条件培养基对绵羊类胚胎干细胞克隆、传代的影响[J]. 畜牧兽医学报, 2009, 40(9): 1327-1332.

BAI Changming;WANG Xinzhuang;YAN Yingying;HE Xiaoe;ZHANG Jingfang . The Effects of Mouse ES Cell Conditioned Medium on Isolation and Cloning of Sheep Embryonic Stemlike Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(9): 1327-1332.

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