山羊iPS细胞诱导及培养体系的优化

doi:10.11843/j.issn.0366-6964.2014.05.009

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (5): 740-749.doi: 10.11843/j.issn.0366-6964.2014.05.009

山羊iPS细胞诱导及培养体系的优化

邱峰龙，左其生，李东，李伟，陈庭锋，韦光辉，李碧春^*

（扬州大学动物科学与技术学院，扬州 225009）

收稿日期:2013-12-27 出版日期:2014-05-23 发布日期:2014-05-23
通讯作者: 李碧春，博士生导师，教授， E-mail：yubcli@yzu.edu.cn
作者简介:邱峰龙（1989-），男，山东潍坊人，硕士生，主要从事动物胚胎工程研究，E-mail：qiufenglongone@126.com
基金资助:
转基因生物新品种培育重大专项（2011ZX08008-003）；国家高技术研究发展计划（2011AA100307-04）；江苏高校优势学科建设工程资助项目

Optimization of Induced and Cultivation System of Goat iPS Cells

QIU Feng-long， ZUO Qi-sheng， LI Dong， LI Wei， CHEN Ting-feng， WEI Guang-hui， LI Bi-chun^*

(College of Animal Science and Technology，Yangzhou University，Yangzhou 225009， China)

Received:2013-12-27 Online:2014-05-23 Published:2014-05-23

摘要/Abstract

摘要：

为了高效、持续的诱导获得并培养山羊诱导性多能干细胞（Induced pluripotent stem cells，iPS），本研究从饲养层和培养液方面进行优化。将分离获得3代以内的小鼠胎儿成纤维细胞（Mouse embryonic fibroblast，MEF）、山羊胎儿成纤维细胞（Goat embryonic fibroblast，GEF）用丝裂霉素C处理后，分别按1×10⁵ mL^-1MEF、1×10⁵mL^-1GEF、5×10⁴ mL^-1MEF+5×10⁴ mL^-1GEF 的密度接种，以高糖DMEM+20%胎牛血清（FBS）和KnockoutDMEM+20% 血清替代物（KSR）为培养液，研究不同饲养层种类和培养液对山羊iPS细胞获得和培养的影响。结果显示，山羊iPS细胞在接种密度为 5×10⁴ mL^-1MEF+5×10⁴ mL^-1GEF 的饲养层上，使用 KnockoutDMEM+20% KSR 组合的培养液，山羊iPS细胞的诱导效率以及消化传代后的克隆形成率均极显著高于其他5组（P＜0.01）。对该组培养的山羊iPS细胞进行碱性磷酸酶（AKP）染色呈阳性；Oct4、SSEA-1、Tra-1-60、Tra-1-81免疫荧光检测呈阳性；RT-PCR检测有Oct4、Sox2、Klf4和Nanog基因的表达；体外能分化形成类胚体。结果表明，将细胞接种在密度为 5×10⁴ mL^-1MEF+5×10⁴ mL^-1GEF 的饲养层上，使用 KnockoutDMEM+20% KSR 的培养液更适合山羊iPS细胞的获得和培养。本试验为山羊iPS细胞的体外研究、临床试验、动物基因组修饰和山羊胚胎干细胞（Embryonic stem cells，ES）的建系奠定基础。

Abstract:

In order to induced and cultured the goat iPS cells efficiently and continuous，the feeder cells and culture conditions were optimized in this study.MEF and GEF cells were inoculated at concentrations of 1×10⁵ mL^-1MEF,1×10⁵ mL^-1GEF and 5×10⁴ mL^-1MEF+5×10⁴mL^-1GEF as the feeder layers of goat iPS cells after they were treated with Mitomycin C within 3 passages，and cultured in DMEM+20%FBS,KnockoutDMEM+20%KSR，observed the different influence.The results showed that the induction efficiency and formation rates after digest of goat iPS cells in 5×10⁴ mL^-1MEF+5×10⁴ mL^-1GEF and Knockout DMEM+20% KSR were more higher than those cultured on the other 5 groups(P＜0.01).Growth states of goat iPS cells were observed in this group and undifferentiated phenotypes were detected，included expression of alkaline phosphatase，and dected the expression of Oct4,Sox2,Klf4,Nanog and cell marker Oct4,SSEA-1,Tra-1-60,Tra-1-81 by RT-PCR.In the condition of feeder layer free，goat iPS cells differentiation were observed in vitro. The results indicated that the acquisition and cultivate of goat iPS cells in 5×10⁴ mL^-1MEF+5×10⁴ mL^-1GEF and KnockoutDMEM+20% KSR were more easily than those cultured on the other 5 groups.This study established a foundation for further study on iPS cell in vitro studies，clinical trials，animal genome modification and goat ES cells line.

中图分类号:

S827
Q343

邱峰龙，左其生，李东，李伟，陈庭锋，韦光辉，李碧春. 山羊iPS细胞诱导及培养体系的优化[J]. 畜牧兽医学报, 2014, 45(5): 740-749.

QIU Feng-long， ZUO Qi-sheng， LI Dong， LI Wei， CHEN Ting-feng， WEI Guang-hui， LI Bi-chun. Optimization of Induced and Cultivation System of Goat iPS Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(5): 740-749.

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山羊iPS细胞诱导及培养体系的优化

Optimization of Induced and Cultivation System of Goat iPS Cells

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