第28期鸡胚原始生殖细胞的分离培养

Isolation and Culture of Primordial Germ Cells of Chicken Embryo at 28th Stage

[目的]进一步优化生殖细胞(PGCs)细胞体外培养条件。[方法]从第28期(5.5 d)鸡胚生殖嵴分离PGCs,将PGCs与性腺基质细胞共培养进行原代培养,比较2种培养温度和3种培养浓度对PGCs原代培养的影响,以及2种饲养层细胞对PGCs传代培养的影响,并通过倒置显微镜下形态学观察、碱性磷酸酶(AKP)和过碘酸希夫反应(PAS)染色方法鉴定PGCs。[结果]培养浓度为2.5×105个/m l和培养温度为37℃时PGCs增殖较多,不易分化,存活能力较强,以第2代鸡胚成纤维细胞作饲养层时传代培养效果较好,获得了PGCs增殖的未分化集落,为后期的细胞标记及移植研究提供了较多数量和较高活力的PGCs。[结论]获得了PGCs原代与传代培养较好的培养体系,为禽类EG细胞系的建立和转基因研究奠定了基础。

[ Objective ] The study aimed to optimize the conditions for culture in vitro of primordial germ cells （PGCs）. [ Method ] PGCs were isolated from the genital ridges of chicken embryos at the 28th stage （5.5 d） , then they were co-cultured with gonad stroma cells for primary culture. The effect of 2 kinds of culture temperatures and 3 kinds of culture concn, on PGCs primary culture and that of 2 kinds of feeder layer cells on PGCs subcuhure were compared. PGCs were identified by morphologic observation under inverted microscope and staining of alkaline phosphatase （AKP） and periodic acid-schiff regent （PAS）. [ Result ] PGCs could proliferate, maintain undifferentiated situation, and had stronger viability when the cell culture eonen, was 2.5 × 10^5/ml and the culture temperature was 37 ℃. The second passage of chicken embryo fibroblast （CEF） as the feeder layer was more suitable for PGCs subculture and got the undifferentiated colony of PGCs proliferation, which afforded the ample PGCs with high viability for the subsequent experiment of PGCs labeling and transplantation. [ Conclusion] The culture system with better primary culture and subculture for PGCs was obtained, which laid the foundation for establishment of avian cell line and its transgenic research.