牛囊胚ICM克隆多能性标记基因与表面标记的研究

doi:10.11843/j.issn.0366-6964.2015.07.009

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (7): 1141-1149.doi: 10.11843/j.issn.0366-6964.2015.07.009

牛囊胚ICM克隆多能性标记基因与表面标记的研究

崔莉莎^1，2，赵学明²，郝海生²，杜卫华²，马友记¹，朱化彬^2*，王宗礼^1*

（1.甘肃农业大学动物科学技术学院，兰州 730070； 2.中国农业科学院北京畜牧兽医研究所，北京 100193）

收稿日期:2015-01-05 出版日期:2015-07-23 发布日期:2015-07-20
通讯作者: 朱化彬，博士，研究员，E-mail：zhuhuabin@caas.cn；王宗礼，博士，研究员，E-mail：Wangzongli@sina.com
作者简介:崔莉莎(1989-)，女，山东菏泽人，硕士生，主要从事动物遗传育种与繁殖方面的研究，E-mail：cuilishaxz@163.com
基金资助:
中国农业科学院北京畜牧兽医研究所家畜胚胎工程与繁殖创新团队（ASTIP-IAS06-2014）；中央级公益性科研院所基本科研业务费专项（2014ywf-yb-2）

The Research of Pluripotency-related Marker Genes and Surface Markers in Bovine ICM Colonies

CUI Li-sha^1，2，ZHAO Xue-ming²，HAO Hai-sheng ²，DU Wei-hua ²，MA You-ji¹，ZHU Hua-bin^2*，WANG Zong-li^1*

（1.College of Animal Science and Technology，Gansu Agricultural University，Lanzhou 730070，China；2.Institute of Animal Science，Chinese Academy of Agricutural Sciences，Beijing 100193，China）

Received:2015-01-05 Online:2015-07-23 Published:2015-07-20

摘要/Abstract

摘要：

本研究旨在探究牛类胚胎干细胞（Embryonic stem cells，ES）的多能性标记基因与表面标记，为优化牛类ES细胞培养条件和相关研究提供依据。利用2i/LIF培养液，通过全胚接种及机械传代法分离培养牛囊胚内细胞团（Inner cell mass，ICM），免疫荧光染色法检测其多能性标记基因与表面标记；qRT-PCR检测免疫磁珠法分选的牛囊胚ICM和滋养层细胞（Trophectoderm cell，TE）的多能性标记基因的差异表达结果。试验成功分离出了牛囊胚ICM克隆，并体外培养至第10代，且各代克隆均呈现出了典型的干细胞形态。结果表明，ICM表面标记SSEA1、SSEA4和TRA-1-60染色为阳性，且多能性标记基因OCT4、SOX2和NANOG在其中均有表达；OCT4、SOX2和NANOG在牛囊胚ICM和TE中的表达存在差异（P<0.05），其中 SOX2 的差异极显著（P<0.01）。综上表明，2i/LIF培养液有助于牛囊胚ICM的培养；SOX2可能成为牛ICM克隆的候选多能性标记基因。

关键词: 牛, ICM, 多能性标记, qRT-PCR

Abstract:

This study was designed to investigate the pluripotency-related markers of bovine ES cells so as to provide a basis for optimizing culture conditions of bovine ES cells and promoting its research progress.The inner cell mass (ICM) were separated from bovine IVF blastocysts，seeded on mouse embryonic fibroblasts cultured in 2i/LIF medium and then passaged mechanically.The pluripotent markers of the ICM-derived colonies were detected at passage 1 and passage 10 by immunostaining.Additionally，the ICM and trophectoderm (TE) isolated from bovine blastocysts by magnetic activated cell sorting were used for qRT-PCR to validate the differences in gene expression between ICM and TE.We have derived ICM colonies from bovine IVF blastocysts and cultured them to passage 10，which displayed typical stem cell morphology and expressed specific markers such as OCT4，SOX2，NANOG，SSEA1，SSEA4 and TRA-1-60.Moreover，the results of qRT-PCR showed that OCT4，SOX2 and NANOG were differentially expressed between ICM and TE，SOX2 showed the most significant difference.In conclusion，2i/LIF culture medium is helpful for the culture of the ICM-derived colonies from bovine IVF blastocysts，and SOX2 is more likely to be a candidate pluripotency-related marker gene of bovine ICM-derived colonies.

Key words: bovine, ICM, pluripotency-related marker, qRT-PCR

中图分类号:

S823.2

崔莉莎，赵学明，郝海生，杜卫华，马友记，朱化彬，王宗礼. 牛囊胚ICM克隆多能性标记基因与表面标记的研究[J]. 畜牧兽医学报, 2015, 46(7): 1141-1149.

CUI Li-sha,ZHAO Xue-ming,HAO Hai-sheng,DU Wei-hua,MA You-ji，ZHU Hua-bin,WANG Zong-li. The Research of Pluripotency-related Marker Genes and Surface Markers in Bovine ICM Colonies[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(7): 1141-1149.

参考文献

［1］KIM E Y，NOH E J，PARK H Y，et al.Establishment of bovine embryonic stem cell lines using a minimized feeder cell drop［J］.Cell Reprogram，2012，14(6)：520-529.
［2］RATHJEN J，LAKE J A，BETTESS M D，et al.Formation of a primitive ectoderm like cell population，EPL cells，from ES cells in response to biologically derived factors［J］.J Cell Sci，1999，112(Pt5)：601-612.
［3］EVANS M J，KAUFMAN M H.Establishment in culture of pluripotential cells from mouse embryos［J］.Nature，1981，292（5819）：154-156.
［4］THOMSON J A，KALISHMAN J，GOLOS T G，et al.Isolation of a primate embryonic stem-cell line［J］.Proc Natl Acad Sci U S A，1995，92(17)：7844-7848.
［5］THOMSON J A，ITSKOVITZ-ELDOR J，SHAPIRO S S，et al.Embryonic stem cell lines derived from human blastocysts［J］.Science，1998，282(5391)：1145-1147.
［6］LI P，TONG C，MEHRIAN-SHAI R，et al.Germline competent embryonic stem cells derived from rat blastocysts［J］.Cell，2008，135(7)：1299-1310.
［7］STICE S L，STRELCHENKO N S，KEEFER C L，et al.Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer［J］.Biol Reprod，1996，54(1)：100-110.
［8］CIBELLI J B，STICE S L，GOLUEKE P J，et al.Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells［J］.Nat Biotechnol，1998，16(7)：642-646.
［9］IWASAKI S，CAMPBELL K H，GALLI C，et al.Production of live calves derived from embryonic stem-like cells aggregated with tetraploid embryos［J］.Biol Reprod，2000，62(2)：470-475.
［10］MITALIPOVA M，BEYHAN Z，FIRST N L.Pluripotency of bovine embryonic cell line derived from precompacting embryos［J］.Cloning，2001，3(2)：59-67.
［11］SAITO S，SAWAI K，UGAI H，et al.Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells［J］.Biochem Biophys Res Commun，2003，309(1)：104-113.
［12］WANG L，DUAN E，SUNG L Y，et al.Generation and characterization of pluripotent stem cells from cloned bovine embryos［J］.Biol Reprod，2005，73(1)：149-155.
［13］CAO S，WANG F，CHEN Z，et al.Isolation and culture of primary bovine embryonic stem cell colonies by a novel method［J］.J Exp Zool A Ecol Genet Physiol，2009，311(5)：368-376.
［14］FURUSAWA T，OHKOSHI K，KIMURA K，et al.Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses［J］.Biol Reprod，2013，89(2)：28.
［15］CONG S，CAO G，LIU D.Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro［J］.Cytotechnology，2014，66(6)：995-1005.
［16］NAGATOMO H，KAGAWA S，KISHI Y，et al.Transcriptional wiring for establishing cell lineage specification at the blastocyst stage in cattle［J］.Biol Reprod，2013，88(6)：158.
［17］MUÑOZ M，RODRÍGUEZ A，FRUTOS C，et al.Conventional pluripotency markers are unspecific for bovine embryonic-derived cell-lines［J］.Theriogenology，2008，69(9)：1159-1164.
［18］LA BLOMBERG，BPVL TELUGU.Twenty years of embryonic stem cell research in farm animals［J］.Reprod Dom Anim，2012，47(Suppl4)：80-85.
［19］OZAWA M，HANSEN P J.A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting［J］.Fertil Steril，2011，95(2)：799-802.
［20］OZAWA M，SAKATANI M，HANKOWSKI K E，et al.Importance of culture conditions during the morula-to-blastocyst period on capacity of inner cell-mass cells of bovine blastocysts for establishment of self-renewing pluripotent cells［J］.Theriogenology，2012，78(6)：1243-1251.
［21］VERMA V，HUANG B，KALLINGGAPPA P K，et al.Dual kinase inhibition promotes pluripotency in finite bovine embryonic cell lines［J］.Stem Cells Dev，2013，22(11)：1028-1042.
［22］BRACKETT B G，OLIPHANT G.Capacitation of rabbit spermatozoa in vitro［J］.Biol Reprod，1975，12（2）：260-274.
［23］ROSENKRANS C F，JR FIRST N L.Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro［J］.J Anim Sci，1994，72(2)：434-437.
［24］GONG G，ROACH M L，JIANG L，et al.Culture conditions and enzymatic passaging of bovine ESC-like cells［J］.Cell Reprogram，2010，12(2)：151-160.
［25］BUEHR M，MEEK S，BLAIR K，et al.Capture of authentic embryonic stem cells from rat blastocysts［J］.Cell，2008，135(7)：1287-1298.
［26］SCHMITTGEN T D，LIVAK K J.Analyzing real-time PCR data by the comparative C (T) method［J］. Nat Protoc，2008，3（6）：1101-1108.
［27］GOOI H C，FEIZI T，KAPADIA A，et al.Stage-specific embryonic antigen involves alpha 1 goes to 3 fucosylated type 2 blood group chains［J］.Nature，1981，292(5819)：156-158.
［28］ANDREWS P W，DAMJANOV I，SIMON D，et al.Pluripotency embryonal carcinoma clones derived from human teratocarcinoma cell line Tera-2：differentiation in vivo and in vitro［J］.Lab Invest，1984，50(2)：147-162.
［29］GOISSIS M D，CIBELLI J B.Functional characterization of SOX2 in bovine preimplantation embryos［J］.Biol Reprod，2014，90(2)：30.
［30］SUMER H，LIU J，MALAVER-ORTEGA L F，et al.NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts［J］.J Anim Sci，2011，89(9)：2708-2716.
［31］YINDEE K J S，JIANG G，KANOK P，et al.Establishment and long-term maintenance of bovine embryonic stem cell lines using mouse and bovine mixed feeder cells and their survival after cryopreservation［J］.Science Asia，2000，26：81-86.
［32］VRANA K E，HIPP J D，GOSS A M，et al.Nonhuman primate parthenogenetic stem cells［J］.Proc Natl Acad Sci U S A，2003，100(suppl 1)：11911-11916.
［33］HARRIS D，HUANG B，OBACK B.Inhibition of MAP2K and GSK3 signaling promotes bovine blastocyst development and epiblast-associated expression of pluripotency factors［J］.Biol Reprod，2013，88(3)：74.
［34］BERG D K，SMITH C S，PEARTON D J，et al.Trophectoderm lineage determination in cattle［J］.Dev Cell，2011，20(2)：244-255.

[1]	韩小红, 何翃闳, 王靖雷, 张慧珠, 马悦, 马进彪, 赵生贤, 余四九, 崔燕, 樊江峰. 牦牛p38MAPK在雌性主要生殖器官中的表达[J]. 畜牧兽医学报, 2019, 50(9): 1802-1812.
[2]	康浩然, 刘重阳, 于勇, 张俊杰, 潘子豪, 姚火春. 2017-2018年我国部分地区牛支原体的分离鉴定及多位点序列分型[J]. 畜牧兽医学报, 2019, 50(9): 1857-1863.
[3]	赵凯, 崔燕, 刘鹏刚, 何俊峰, 宋亮丽, 廖博, 杨晓晴. IL-17和HSP90在不同牛肝中的定位和定量研究[J]. 畜牧兽医学报, 2019, 50(9): 1897-1903.
[4]	杨涛, 许厚强, 陈伟, 周迪, 王圆圆, 朱晓锋. 牛MSTN启动子与MEF2C转录因子相互作用的研究[J]. 畜牧兽医学报, 2019, 50(8): 1567-1575.
[5]	罗芳, 郭延生, 马志远, 卢旺银, 李刚, 陶金忠. 基于UPLC-Q-TOF MS代谢组学技术筛选表征西门塔尔种公牛精子活力的候选生物标志物[J]. 畜牧兽医学报, 2019, 50(8): 1596-1606.
[6]	温东旭, 张雷, 索朗斯珠, 牛家强, 王玉恒, 贡嘎, 旦巴次仁, 席广银, 郭敏, 徐业芬. 牦牛Nramp1基因mRNA组织表达谱及其相关miRNAs初步研究[J]. 畜牧兽医学报, 2019, 50(8): 1576-1586.
[7]	张芮琪, 张莹利, 蒋明, 徐越, 徐永平, 卿素珠. 成纤维生长因子4在妊娠早期牛胎盘组织的分布及表达特点[J]. 畜牧兽医学报, 2019, 50(8): 1685-1693.
[8]	杨涛, 许厚强, 陈伟, 周迪, 王圆圆, 朱晓锋. 牛MSTN启动子与MEF2C转录因子相互作用的研究[J]. 畜牧兽医学报, 2019, 50(8): 1567-1575.
[9]	温东旭, 张雷, 索朗斯珠, 牛家强, 王玉恒, 贡嘎, 旦巴次仁, 席广银, 郭敏, 徐业芬. 牦牛Nramp1基因mRNA组织表达谱及其相关miRNAs初步研究[J]. 畜牧兽医学报, 2019, 50(8): 1576-1586.
[10]	罗芳, 郭延生, 马志远, 卢旺银, 李刚, 陶金忠. 基于UPLC-Q-TOF MS代谢组学技术筛选表征西门塔尔种公牛精子活力的候选生物标志物[J]. 畜牧兽医学报, 2019, 50(8): 1596-1606.
[11]	张芮琪, 张莹利, 蒋明, 徐越, 徐永平, 卿素珠. 成纤维生长因子4在妊娠早期牛胎盘组织的分布及表达特点[J]. 畜牧兽医学报, 2019, 50(8): 1685-1693.
[12]	刘江静, 陈晓丽, 田宏志, 路永强, 王栋. 返情母牛活动量影响因素分析[J]. 畜牧兽医学报, 2019, 50(7): 1405-1411.
[13]	俞演, 古玉, 王承东, 胡静, 杨瑷宁, 杨乔, 李德生, 张和民, 邓林华, 凌珊珊, 左之才, 彭广能, 钟志军, 马晓平. 石膏样小孢子菌感染小鼠的Dectin-1、TLR-2和TLR-4及细胞因子动态变化[J]. 畜牧兽医学报, 2019, 50(7): 1449-1457.
[14]	朱斌, 刘小倩, 刘颖, 纪晓霞, 张源淑. 血管紧张素转化酶2对Ang Ⅱ诱导的牛乳腺上皮细胞炎症损伤的缓解作用[J]. 畜牧兽医学报, 2019, 50(7): 1458-1465.
[15]	刘江静, 陈晓丽, 田宏志, 路永强, 王栋. 返情母牛活动量影响因素分析[J]. 畜牧兽医学报, 2019, 50(7): 1405-1411.

牛囊胚ICM克隆多能性标记基因与表面标记的研究

The Research of Pluripotency-related Marker Genes and Surface Markers in Bovine ICM Colonies

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价