饲养层密度对兔类ES细胞分离培养的影响

doi:10.11843/j.issn.0366-6964.2013.07.009

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (7): 1051-1056.doi: 10.11843/j.issn.0366-6964.2013.07.009

饲养层密度对兔类ES细胞分离培养的影响

李小佳，张君涛，徐秋勤，王鸽，曹书郡，王新庄，张志平^*

（河南农业大学牧医工程学院，郑州 450002）

收稿日期:2013-01-16 出版日期:2013-07-23 发布日期:2013-07-23
通讯作者: 张志平，博士，主要从事胚胎工程与干细胞研究，E-mail：zzping95@163.com
作者简介:李小佳(1985-)，女，河南新密人，硕士生，主要从事兔胚胎干细胞诱导分化研究，E-mail: 1536957524@qq.com
基金资助:
国家自然科学基金（31001096）

Effect of Densities of Feeder Layers for Isolation and Culturing of Rabbit ES-like Cells

LI Xiao-jia, ZHANG Jun-tao, XU Qiu-qin, WANG Ge, CAO Shu-jun, WANG Xin-zhuang, ZHANG Zhi-ping^*

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

Received:2013-01-16 Online:2013-07-23 Published:2013-07-23

摘要/Abstract

摘要：

探讨饲养层细胞接种密度对兔类ES细胞分离培养影响，筛选适合兔类ES细胞分离培养最适饲养层密度。将兔囊胚分别接种到密度为10×10³、20×10³和40×10³ 个·cm^-2的小鼠胚胎成纤维细胞(Mouse embryonic fibroblast cell, MEF)饲养层上，观察兔类ES细胞贴壁和克隆生长情况，并对所得兔类ES细胞进行碱性磷酸酶和Oct-4检测。结果表明，接种于密度为20×10³ 个·cm^-2饲养层上的囊胚贴壁率（82.5%）、内细胞团（Inner cell mass, ICM）形成率（67.5%）高于接种于密度为40×10³ 个·cm^-2的饲养层上囊胚贴壁率(80.0%）、内细胞团（Inner cell mass ，ICM)形成率（65.7%），差异不显著（P>0.05）；显著高于（P<0.05）接种于密度为10×10³ 个·cm^-2饲养层上的囊胚贴壁率（55.6%）、ICM形成率（44.4%）。密度为20×10³ 个·cm^-2饲养层上的兔类ES细胞F1、F2代细胞克隆率均显著高于其他两组（P<0.05），最高已传至20代；获得的兔类ES细胞经碱性磷酸酶染色及表面抗原Oct-4表达检测均呈阳性。密度为20×10³ 个·cm^-2的MEF饲养层可更好支持兔类ES细胞体外培养。

Abstract:

To explore the effects of different densities of feeder layer cells on isolation and culturing of rabbit ES-like cells and select the better culture system. Rabbit blastulas were cultured at different densities(10×10³, 20×10³, and 40×10³·cm^-2) of mouse embryonic fibroblast cell (MEF) as feeder layer, rabbit ES-like cell adherence and growth behavior were observed and express pluripotency markers were detected, such as AKP and Oct-4. The results showed that the attachment rate of blastulas and ICM formation rate at a density of 20×103·cm-2 feeder layer were higher than those at 40×10³·cm^-2 (82.5% vs 80.0%; 67.5% vs 65.7%, respectively), but the two groups were not significantly different (P>0.05). While stem cells cultured at a density of 20×10³·cm^-2 feeder layer were significantly higher than at 10×10³·cm^-2 feeder layer (82.5% vs 55.6%; 67.5% vs 44.4%, respectively) (P<0.05). The cell clone rate of F1、F2 generation of rabbit ES-like cells at a density of 20×10³·cm^-2 feeder layer was significantly higher than other two groups (P<0.05), and the stem cells obtained were up to the 20th passages and expression of AKP and Oct-4 were positive. In conclusion, the MEF feeder layer at a density of 20×10³·cm^-2 may be better suitable for rabbit ES-like cells culture in vitro.

中图分类号:

S829.1
Q813

李小佳，张君涛，徐秋勤，王鸽，曹书郡，王新庄，张志平. 饲养层密度对兔类ES细胞分离培养的影响[J]. 畜牧兽医学报, 2013, 44(7): 1051-1056.

LI Xiao-jia, ZHANG Jun-tao, XU Qiu-qin, WANG Ge, CAO Shu-jun, WANG Xin-zhuang, ZHANG Zhi-ping. Effect of Densities of Feeder Layers for Isolation and Culturing of Rabbit ES-like Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(7): 1051-1056.

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饲养层密度对兔类ES细胞分离培养的影响

Effect of Densities of Feeder Layers for Isolation and Culturing of Rabbit ES-like Cells

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