昆明小鼠胚胎干细胞培养体系的优化

畜牧兽医学报

昆明小鼠胚胎干细胞培养体系的优化

任卫青，张志平，许晓婷，邓立新，李小佳，吕婧玉，翟明胜，王新庄^*

（河南农业大学牧医工程学院，郑州 450002）

收稿日期:2012-02-28 出版日期:2013-02-23 发布日期:2013-02-23
通讯作者: 王新庄(1963-)，男, 博士, 教授，E-mail: wangxinzhuang@yahoo.com.cn
作者简介:任卫青(1986-)，女，河南林州人，硕士，主要从事动物细胞与胚胎工程方面研究，E-mail: guoping6767@126.com
基金资助:
河南省基础与前沿技术研究计划（092300410081）；国家肉牛、牦牛产业体系（30300324）

Optimization of Culture System of Embryonic Stem Cells from Kunming Mouse

REN Wei-qing, ZHANG Zhi-ping, XU Xiao-ting, DENG Li-xin, LI Xiao-jia, LV Jing-yu, ZHAI Ming-sheng, WANG Xin-zhuang^*

(College of Animal Science and Veterinary Medicine, Henan Agricultural University,Zhengzhou 450002, China)

Received:2012-02-28 Online:2013-02-23 Published:2013-02-23

摘要/Abstract

摘要：

为了更高效地分离昆明小鼠胚胎干细胞，本研究从饲养层、胚胎发育阶段和培养液方面进行优化。将3代以内的小鼠胎儿成纤维细胞（MEF）用丝裂霉素C处理后，分别按1×10⁴、1×10⁵、1×10⁶·mL^-1密度接种，以H-DMEM+15%KSR+LIF为培养液，观察不同密度饲养层对昆明小鼠胚胎干细胞(ES细胞)生长的影响，并研究胚胎发育阶段和培养液中分别添加干细胞生长因子(SCF)、SCF +胰岛素对昆明小鼠ES细胞分离克隆的影响。结果显示，胚胎在密度为1×10⁵·mL^-1的饲养层上， F1代和F2代ES细胞克隆形成率均显著高于其他2组(P<0.05)。囊胚的F2代ES细胞克隆形成率显著高于桑椹胚(P<0.05)，培养液中添加SCF显著提高昆明小鼠胚胎贴壁率(P<0.05)，同时添加SCF和胰岛素得到昆明小鼠最高胚胎贴壁率及F1、F2代ES细胞克隆形成率。所分离的ES细胞显示AKP染色强阳性，Oct-4、SSEA-1的免疫荧光检测阳性，具有 ES细胞的特点。由此认为，发育至囊胚的胚胎在MEF密度为1×10⁵·mL^-1上，培养液中同时添加SCF和胰岛素更适合昆明小鼠ES细胞的分离培养。

Abstract:

In order to isolate the ES cells from Kunming mouse efficiently, the feeder cells, developmental stage of embryos and culture conditions were optimized in the study. Mouse embryonic fibroblast (MEF) were inoculated at concentrations of 1×10⁴, 1×10⁵ and 1×10⁶·mL^-1 as the feeder layers of ES cells after they were treated with Mitomycin C within 3 passages, and cultured in H-DMEM+15%KSR+LIF, the growth of the ES cells were observed. The effects of developmental stages of embryos and added SCF, SCF+insulin in culture medium on growth of Kunming mouse ES cells were investigated. The results showed that the formation rates of the 1^st and 2^nd passage ES cells in 1×10⁵·mL^-1 MEF were higher than those cultured on the other 2 groups(P<0.05). The 2^nd passage ES cells colonies formation rate of blastocysts were higher than that of compacted morula(P<0.05). It had improved the attachment rate was significantly enhanced(P<0.05) when the culture medium added with SCF, and the highest attachment rate, 1^st and 2^nd passage ES cells colonies formation rate were got when the culture medium added with SCF and insulin (P<0.05). The isolated ES cells, which were positive for AKP staining and immunofluorescence against antigens of Oct-4 and SSEA-1, had a series of characters of mice ES cells. These results suggest that the blastocysts on the MEF with a density of 1×10⁵·mL^-1 and addition with SCF and insulin in the culture medium is more suitable for the isolation and passage of Kunming mouse ES cells.

中图分类号:

任卫青，张志平，许晓婷，邓立新，李小佳，吕婧玉，翟明胜，王新庄. 昆明小鼠胚胎干细胞培养体系的优化[J]. 畜牧兽医学报, doi: .

REN Wei-qing, ZHANG Zhi-ping, XU Xiao-ting, DENG Li-xin, LI Xiao-jia, LV Jing-yu, ZHAI Ming-sheng, WANG Xin-zhuang. Optimization of Culture System of Embryonic Stem Cells from Kunming Mouse[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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