解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (3 ): 397-402.doi: 10.3969/j.issn.0529-1356.2013.03.021

• 组织学胚胎学发育生物学 • 上一篇    下一篇

小鼠胚胎生殖细胞的建立及其印记状态

胡静1,2  赵巧湜1  古艳丽1  白光宇1  吴稀1  雷蕾*    

  1. 1.哈尔滨医科大学组织学与胚胎学教研室，哈尔滨 150081;2.牡丹江医学院组织学与胚胎学教研室，黑龙江 牡丹江 157011
  • 收稿日期:2012-06-08 修回日期:2012-08-07 出版日期:2013-06-06 发布日期:2013-07-16
  • 通讯作者: 雷 蕾 E-mail:Leil086@yahoo.com.cn
  • 基金资助:

    黑龙江省青年专项基金项目（QC2011C056）；黑龙江省研究生创新科研项目（YJSCX2011-321HLJ）;其他(不属于以上基金类别的请自行输入下框)

Establishment of a mouse embryonic germ cell line and preliminary study of the expression of imprinted genes

HU Jing 1,2  ZHAO Qiao-shi1  GU Yan-li1  BAI Guang-yu1  WU Xi1  LEI Lei 1*   

  1. 1.Department of Histology and Embryology, Harbin Medical University, Harbin 150081, China;
    2. Department of Histology and Embryology,Mudanjiang Medical University,  Heilongjiang Mudanjiang 157011,  China
  • Received:2012-06-08 Revised:2012-08-07 Online:2013-06-06 Published:2013-07-16

摘要：

目的 成功建立小鼠胚胎生殖细胞（EGCs）系，并初步分析小鼠胚胎生殖细胞的印记状态。方法 建立交配后12.5d（12.5dpc）原始生
殖细胞 (PGCs）来源的小鼠EGCs，通过碱性磷酸酶（AKP）染色、免疫荧光细胞化学、体内分化及体外分化等方法检测EGCs的多能性，并以小鼠
胚胎干细胞（ESCs）为对照，应用Real-time PCR检测EGCs中与发育相关的Ins2、Lgf2、H19、Lgf2r等11个父源与母源印记基因的表达情况。结果
成功建立小鼠EG细胞系，EGCs克隆AKP染色显示有高水平的AKP活性，免疫荧光细胞化学方法显示克隆表达小鼠ESCs多能性标记物Oct4及细胞表面
标记SSEA-1。核型分析检测显示，小鼠EGCs为正常的40条染色体，体内可分化出3个胚层来源的组织，说明小鼠EGCs具有多能性；Real-time PCR
结果显示EGCs的印记基因表达量显著高于ESCs。结论 12.5dpc PGC来源的EGCs的印记基因处于擦除状态。

关键词: 胚胎生殖细胞, 印记基因, 实时定量聚合酶链反应, 小鼠

Abstract:

Objective  To establish a mouse embryonic germ cell (EGCs) line and to detect the expression of imprinted genes
in order to provide basic information for further study and application of embryonic germ cells. Methods EGCs were isolate
from primordial germ cells collected from the genital ridge of 12.5 days postcoitum (dpc). The pluripotent characteristics of
the established EGCs were detected by alkaline phophatase (AKP) staining, immunofluorescent detection of mouse embryonic stem
cells (ESCs) surface antigens, and cell differentiations in vivo. The expressions of several patrilineal and matrilineal
imprinted genes, such as Ins2, Lgf2, H19, Lgf2r and so forth, were also detected by quantitative reverse transcriptase-polymerase
chain reaction in both EGCs and ESCs. Results The EGCs showed positive for alkaline phosphatase. The pluripotency marker Oct4
and the cell surface marker SSEA-1 were also shown in EGCs cells. Karyotype analysis indicted that EGCs had normal 40 chromosomes,
and differentiated into the tissues presenting three germinal layers derivations in vivo, suggesting that embryonic germ cells
had pluripotent characteristics. Real-time PCR showed that the expression levels of imprinted genes in EGCs were significantly
highter compared with those in ESCs. Conclusion The genomic imprinting memories in EGCs generated from primordial germ cells
which collected from the genital ridge of 12.5 dpc are completely erased.

Key words: Embryonic germ cell, Imprinted gene, Real-time PCR, Mouse

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