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  

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

作　　者：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] 

机构地区：[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

出　　处：《中国畜牧兽医》2007年第3期55-59,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金资助(30170678)

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+0.1mol/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。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 media 1. 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 subcuhured after slow speed cryopreservation and the second passage PGCs subcuhured after vitrificated cryopreservation were positive to PAS and AKP staining and still kept intact karyotypes.

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 

分 类 号：Q813.7[生物学—生物工程]