›› 2007, Vol. 34 ›› Issue (3): 55-59.

• 遗传繁育 • 上一篇    下一篇

鸡胚19期原始生殖细胞慢速冷冻和玻璃化冷冻保存的研究

韩威1，李碧春1，朱云芬1，秦洁1，周冠月1，陈国宏1，张学余2
   

  1. 1.扬州大学动物科学与技术学院，扬州 225009；2.中国农业科学院家禽研究所，扬州 225003
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-03-20 发布日期:2007-03-20

Study on the Slow Speed Cryopreservation and Vitrificated Cryopreservation for Chicken PGCs at Stage 19

HAN Wei1，LI Bichun1，ZHU Yunfen1，QIN Jie1，ZHOU Guanyue1，
CHEN Guohong1，ZHANG Xueyu2   

  1. 1.College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; 2.Poultry Institute, Chinese Academy of Agricultural Science, Yangzhou 225003, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2007-03-20 Published:2007-03-20

摘要： 本研究对发育至第19期的鸡胚性腺原始生殖细胞(primordial germ cells，PGCs)分离提纯后，在DMEM中添加冷冻保护剂DMSO（二甲基亚砜）、EG（乙二醇）、蔗糖、PVP（聚乙烯吡咯烷酮）分别组成6种慢速冷冻保护液和6种玻璃化冷冻保护液，进行超低温冷冻保存。复苏后苔盼蓝染色测定细胞存活率，体外接种培养、传代。结果：①慢速冷冻保存中，PGCs在慢速冷冻液V（10％EG+10% FBS+0.1 mol/L蔗糖）条件下复苏后存活率最高（92.20%），且与慢速冷冻液I（10％ DMSO+10% FBS）存活率之间差异显著（P<0.05）。②玻璃化冷冻保存中，PGCs在玻璃化冷冻液I（10% DMSO+10% EG+20% FBS+10% PVP）条件下复苏后存活率最高（84.15%），且与其余5种玻璃化冷冻液下复苏后存活率之间差异均极显著（P<0.01）。③培养传至第3代的慢速冷冻复苏后PGCs细胞和培养传至第2代的玻璃化冷冻复苏后PGCs细胞，PAS染色、AKP染色呈阳性并保持完整的二倍体核型。

关键词: 鸡; 原始生殖细胞; 慢速冷冻保存; 玻璃化冷冻保存; 培养传代

Abstract: This paper focused on isolated primordial germ cells (PGCs) from gonads at stage 19 by Ficoll densitygradient centrifugation. Then, the PGCs were froze respectively in six kinds of freezing medias under slow speed cryopreservation process and vitrification process. The viability of the frozenthawed PGCs was measured by the trypan blue exclusion method. The results showed: For the slow speed cryopreservation, the viability of the frozenthawed PGCs under freezing media V was the highest, and showed significant difference（P<0.05）when compared to that of freezing mediaⅠ.For the vitrificated cryopreservation, the viability of the frozenthawed PGCs under freezing media I was the highest,and showed very significant difference（P<0.01）when compared to that of other five freezing medias. The third passage PGCs subcultured after slow speed cryopreservation and the second passage PGCs subcultured after vitrificated cryopreservation were positive to PAS and AKP staining and still kept intact karyotypes. 

Key words: chicken; PGCs; slow speed cryopreservation; vitrificated cryopreserration; subculture

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