摘要
采用Ficoll密度梯度离心法和EDTA 胰酶酶解法两种方法 ,分别提取第 14期 (孵化 5 3h)血液、第 19期 (孵化 72h)和第 2 8期 (孵化 132h)生殖腺中的PGCs,以比较两种分离方法对 3个发育时期的鸡胚原始生殖细胞在相同体外培养条件下存活时间的差异。结果发现 ,两种分离方法均能分离到一定数量的原始生殖细胞 ,但是酶解法分离到的原始生殖细胞的相对数量较Ficoll密度梯度离心法的多 ,存活时间较长 ,是一种适宜的分离方法 ;对鸡胚发育第 14、 19、 2 8期 3个时期提取的原始生殖细胞进行体外培养 ,存活时间分别为 :72h、 88h和 80h ,三者之间差异显著。结果表明 :在鸡胚孵化的第 19期 ,因原始生殖细胞大量聚集在肢体后端的生殖嵴原基处 ,因而较容易收集 ,体外培养较为适宜 。
We compared both Ficoll density gradient centrifugation and EDTA trypsin collection methods for isolating primordial germ cells (PGCs) from chicken embryos at stage 14,19 and 28, cultured the PGCs in TCM 199 medium containing 10% FCS without any growth factor under 38 5℃, 5%CO 2 and saturation moisture to compare the survival time in vitro . The results showed that the survival duration of the PGCs at stage 14 was 72 h, stage19 was 88 h, and stage 28 was 80 h. The number of PGCs, and the viable PGCs with the EDTA trypsin treatment were more (2 71×10 4, 65 12%) than that of Ficoll density gradient centrifugation(1 78×10 4,45 24%) at stages 19 and 28 Comparison of results of culture of PGCs in vitro at those three development stages indicated that a large amount of PGCs had aggregated in the genital ridge at stage 19 made it convenient for isolation. This stage was suitable for isolation of PGCs from early chicken embryos using EDTA trypsin treatment.
出处
《动物学报》
SCIE
CAS
CSCD
北大核心
2003年第6期835-842,共8页
ACTA ZOOLOGICA SINICA
基金
国家自然科学基金资助 (3 0 170 678)~~
关键词
鸡
原始生殖细胞
分离
存活时间
Chicken, Primordial germ cells (PGCs),Isolation, Survival duration
