鸡胚原始生殖细胞的冷冻保存和体外培养  

Cryopreservation and Culture of Chicken Primordial Germ Cells at Stage 19

作　　者：肖小珺[1] 秦洁[1] 蔡琳琳[1] 李碧春[1] 

机构地区：[1]扬州大学畜牧兽医学院

出　　处：《中国兽医学报》2005年第3期316-319,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30170678)

摘　　要：采用Ficoll密度梯度离心,提取第19期(孵化72h)鸡受精蛋性腺中的原始生殖细胞(primordialgermcells,PGCs),用不同的冷冻保护液对PGCs采用提纯后直接冷冻保存和在培养体系中培养24h后再进行冷冻保存,7d后复苏,用台盼蓝染色检测其存活率,并用PAS染色对冷冻后的PGCs进行特异性鉴定。复苏后的PGCs在培养体系中培养5d后,更换为培养体系继续培养,进行体外分化试验。结果如下分离后直接进行冷冻保存的PGCs复苏后存活率最高为(85.93±1.24)%;复苏后的PGCs在培养体系中培养48h后进行了PAS染色鉴定,结果PGCs呈PAS阳性(细胞核不着色,细胞质呈红色),表明PGCs的染色特性没有因冷冻保存和体外培养而发生改变;复苏后的PGCs在培养体系中培养可形成细胞克隆,当把PGCs更换到培养体系培养5d后,PGCs克隆有的仍保持克隆状态,但体积缩小,克隆中细胞排列紧密,颜色较深,有的克隆由鸟巢状变为不规则形,且在克隆周围形成类神经细胞样细胞。而经体外培养后再进行冷冻保存的PGCs,复苏后存活率最高为(42.26±1.36)%。Primordial germ cells were isolated from gonads at stage 19 by Ficoll density gradient centrifugation.The isolated PGCs were cultured for 24 hours in vitro and cryopreseved in different freezing media.The vitality of the frozen-thawed PGCs was determined by the trypan blue exclusion method.PAS stain were used to identify the characteristic of the PGCs.The PGCs were cultured in culture system Ⅱ and then cultured in culture system Ⅰ to let the PGCs differenciation.The Result showed:The highest vitality is 85.93±1.24 when the PGCs cryopreservated directly after isolated.The PGCs can form colonies when cultured in culture system Ⅱ for 5 h.When the PGCs colony cultured in culture system Ⅰ,some of them disappeared,some of them became small and some differenticated into neuron-like cells.The highest vitality of the PGCs is 42.26±1.36 when cultured for 24 hours in culture system Ⅱ before cryopreservation.

关 键 词：原始生殖细胞 冷冻保存 体外培养 PGCS 培养体系 鸡胚 密度梯度离心 冷冻保护液 台盼蓝染色 特异性鉴定 存活率 PAS 体外分化 染色鉴定 染色特性 细胞克隆 神经细胞 复苏 受精蛋 细胞核 细胞质 不规则 孵化 

分 类 号：S814.8[农业科学—畜牧学] S823.34[农业科学—畜牧兽医]