畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (8): 1333-1340.doi: 10.11843/j.issn.0366-6964.2015.08.008

• 遗传繁育 • 上一篇    下一篇

悬滴培养法促进鸡胚胎干细胞形成类胚体

张蕾1,2,王宵燕1,左其生1,路镇宇1,王飞1,纪艳芹1,王颖洁1,张亚妮1*,李碧春1*   

  1. (1.扬州大学动物科学与技术学院 江苏省动物遗传繁育与分子设计重点实验室,扬州 225009;2.江苏农牧科技职业学院,泰州 225300)
  • 收稿日期:2014-12-11 出版日期:2015-08-23 发布日期:2015-08-23
  • 通讯作者: 张亚妮,讲师,博士,主要从事动物遗传工程研究;李碧春,教授,博士,主要从事动物胚胎工程与遗传工程研究,E-mail:yubcli@yzu.edu.cn
  • 作者简介:张蕾(1987-),女,江苏常州人,博士,主要从事胚胎干细胞体外诱导研究,E-mail:leizhang17@sina.com
  • 基金资助:

    国家自然科学基金(31272429;31472087);江苏省研究生科研创新基金(CXZZ13_0909)

Embryoid Body Formation from Chicken Embryonic Stem Cells through Suspension Culture

ZHANG Lei1,2,WANG Xiao-yan1,ZUO Qi-sheng1,LU Zhen-yu1,WANG Fei1,JI Yan-qin1,WANG Ying-jie1,ZHANG Ya-ni1* ,LI Bi-chun1*   

  1. (1.Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province,College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China;2.Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China)
  • Received:2014-12-11 Online:2015-08-23 Published:2015-08-23

摘要:

本研究旨在通过悬浮培养法培养鸡胚胎干细胞(Embryonic stem cells,ESCs),摸索鸡胚胎干细胞形成类胚体(Embryoid body,EB)的最佳方案,以期提高鸡胚胎干细胞体外诱导效率。 将鸡ESCs培养传代至第3代,采用1×104、3×104和6×104 mL-13个细胞浓度,使用悬滴培养后,观察类胚体的形成效率。对悬滴培养形成的类胚体进行形态学观察,qRT-PCR检测干细胞标记基因的表达,免疫细胞化学检测相关蛋白的表达,并进行类胚体自分化检测和核型分析。3×104 mL-1细胞浓度生成的类胚体数量最高,单个视野下可观察到约267个类胚体,且形成的类胚体大小均一,呈圆球状。qRT-PCR检测结果表明,在类胚体形成48 h内,干细胞标记基因NanogSox2、Oct4和C-kit仍维持表达。免疫细胞化学检测显示,该方法形成的类胚体表面抗原检测NanogSSEA-1呈阳性。自分化检测三胚层分化标记基因SOX17、SMATUJ-1呈阳性。核型分析显示,悬滴培养形成的类胚体具有并保持正常的核型。鸡ESCs悬滴培养形成类胚体的适宜细胞浓度为3×104 mL-1,且形成的类胚体可分化成3个胚层,用于体外定向诱导过程。本研究建立了鸡ESCs体外悬滴培养形成类胚体的培养体系,为后期信号通路的体外验证提供试验基础。

Abstract:

This experiment was conducted to explore the best suspension culture system for embryoid body (EB) formation from chicken embryonic stem cells (ESCs),and to improve the induction efficiency of chicken ESCs in vitro.Pure clones from the third generation of chicken ESCs was collected,re-suspensed the cells into 3 cell concentrations:1×104,3×104 and 6×104 mL-1 to make suspension culture.Morphology changes of EB were observed,qRT-PCR and immunofluorescence methods were used to detect the marker genes’s expression,self-differentiation and karyotype analysis were made to make full test of EB.The results showed that,the amount of EB with 3×104 mL-1 cell concentration was higher than the other groups,and the number of EB reached 267 in one single view with the same spherical shape.The stem cell marker genes Nanog,Sox2,Oct4 and C-kit remained expression with 48 h of EB formation,and stem cell surface specific antigen (Nanog and SSEA-1) detection was positive.After self-differentiation of EB,3 embryonic germ layers specific antigen (SOX17, SMA and TUJ-1) detection all showed positive.Karyotype analysis showed that the formatted EB maintained normal karyotype.The results indicated that the suitable concentration for chicken ESCs form the EB through suspension culture was 3×104 mL-1,and the formatted EB had viability to provide experimental foundation for chicken ESCs induction in vitro.

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