解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (6 ): 812-818.doi: 10.3969/j.issn.0529-1356.2013.06.015

• 组织学胚胎学发育生物学 • 上一篇    下一篇

兔抗小鼠胚胎干细胞免疫血清对8-细胞胚胎发育的影响

胡春超1 崔利芳1 王鹏博1 王棉娟2 邸科前3 李相运1*   

  1. 1. 河北农业大学动物科技学院胚胎工程实验室; 2.河北农业大学校医院内科，河北 保定 071000;3. 河北大学实验动物中心，河北 保定 071000
  • 收稿日期:2012-11-19 修回日期:2013-06-18 出版日期:2013-12-06 发布日期:2013-12-06
  • 通讯作者: 李相运 E-mail:lixiangyun@aliyun.com
  • 基金资助:

    省自然科学基金资助项目

Effects of rabbit antiserum on mouse embryonic stem cells at 8-cell embryo development stage

HU Chun-chao1 CUI Li-fang1 WANG Peng-bo1 WANG Mian-juan2 DI Ke-qian3 LI Xiang-yun 1*   

  1. 1. Laboratroy of Embryo Engineering, Faculty of Animal Science and Technology, Agricultural University of Hebei; 2. Department of Internal Medicine, School Hospital of Agricultural University of Hebei, Hebei Baoding 071000, China;3. Laboratory Animal Centre, Faculty of Medicine, Hebei University, Hebei Baoding 071000, China)
  • Received:2012-11-19 Revised:2013-06-18 Online:2013-12-06 Published:2013-12-06
  • Contact: LI Xiang-yun E-mail:lixiangyun@aliyun.com

摘要：

目的 制备兔抗小鼠胚胎干细胞免疫血清，观察其对小鼠胚胎发育的影响。方法 用小鼠胚胎培养基稀释兔抗血清，分别稀释至50倍、100倍、200倍，用其对小鼠8-细胞胚胎进行培养，通过胚胎致密化、囊胚率、胚胎体外贴壁生长、细胞计数、胚胎移植和免疫荧光染色观察胚胎发育状况。 结果 形态学上50倍稀释的抗血清能够抑制8细胞胚胎的发育甚至使其死亡；100倍稀释的抗血清能够延迟8细胞胚胎的致密化，并使得胚胎在囊胚化过程中出现空泡化的现象；200倍稀释的抗血清作用不明显。未作免疫处理的兔血清与空白对照组差异不显著。100倍稀释的抗血清能够显著抑制囊胚内细胞团（ICM）的发育、囊胚细胞分布紊乱、胚胎细胞数目减少以及囊胚畸形率增加。虽然囊胚的碱性磷酸酶和Oct4均呈阳性，但经胚胎移植后都不能正常发育。 结论 兔抗小鼠胚胎干细胞免疫血清能够抑制小鼠胚胎致密化过程和囊胚内细胞团的发育，使囊胚出现空泡化，细胞分布紊乱，抑制胚胎着床。

关键词: 兔抗血清, 胚胎干细胞, 内细胞团, 致密化, 体外培养, 小鼠

Abstract:

Objective Mouse 8-cell embryos were cultured in KSOM media with rabbit antiserum to mouse embryonic stem (ES) cells, and the effect of the antiserum on mouse embryos was observed. Methods The antiserum was diluted to 1/50, 1/100 and 1/200 by KSOM medium, respectively. Results The result of morphology changes showed that the embryonic development was inhibited or even stopped in the 1/50 dilution group, while the compaction was blocked, the embryos showed fluid vacuoles in the blastula stage in the 1/100 dilution group; there was no obvious effect in the 1/200 dilution group. The treatment group with normal rabbit serum did not appear differences from the control group. Our data indicated that the compaction was blocked by antiserum; when the compacted embryos were exposed in the antiserum, decompaction occurred; prolonged decompaction beyond the 32-cell embryo resulted in an increasing proportion of malformed blastocysts. The embryo development was further tested in the 1/100 dilution group by compaction rate, blastocyst rate, inner cell mass (ICM) outgrowth rate, cell counting, embryo transplantation and fluorescent staining. The result showed that the 1/100 dilution antiserum inhibited ICM formation, decreased embryonic cell number, and creased abnormal blastocyst rate. The treated blastocysts were transferred into uterine horns and did not develop to term. Conclusion Our data suggests that the rabbit antiserum to mouse ES cells inhibits embryo compaction, ICM formation and postimplantation development.

Key words: Rabbit antiserum, Embryonic stem cell, Inner cell mass, Compaction, Culture in vitro, Mouse