水牛PGC和前精原细胞的体外培养

Culture of buffalo PGCs and prespermatogonia in vitro

分别对取自50～95日龄水牛胎儿的原生殖细胞和前精原细胞进行体外培养，观察其生物学行为，并检测其碱性磷酸酶（AP）活性和Oct-4蛋白特性，探讨利用这些生殖细胞建立干细胞系的可行性和检测方法。结果表明：水牛原生殖细胞及前精原细胞分别在体外培养时，均能形成细胞克隆；克隆与周围细胞分界明显，但克隆中细胞相互间界限不清；部分克隆有分隔现象，形如多个克隆共同组成一个大克隆；细胞克隆均至少能培养4代以上；原生殖细胞和前精原细胞及其来源的细胞克隆均呈AP阴性和Oct-4蛋白阴性，其中部分克隆表现为AP假阳性。研究结果显示：水牛原生殖细胞和前精原细胞均可用于建立千细胞系；体外培养时，AP活性和Oct-4蛋白不适宜用来检测这些细胞及其来源的细胞克隆。

The primordial germ cells （PGC） and prespermatogonia of 50 -95 -day- old buffalo fetuses were cultured in vitro and their alkaline phosphatase （AP） activity and Oct -4 protein were detected respectively in order to study the possibility of establishing stem cell lines from them and the detecting methods of them. When the PGC and prespermatogonia of buffalo were cultured in vitro respectively, many cell colonies with various sizes and shapes formed in the culture system. These germ cell colonies were tightly packed with indistinguishable cell -to -cell boundaries, but had a distinct colony boundary from the surrounding feeder layers. Some cell colonies showed compartmentations in them, just like a large colony consisting of several small colonies. These cell colonies could be cultured at least up to 4 passages. The PGCs and prespermatogonia and the cell colonies derived from them displayed AP negative and Oct -4 protein negative, of which some colonies displayed AP pseudo -positive. The results of present study indicate that： （ 1 ） both of the PGCs and prespermatogonia of buffalo embryos/fatuses can be employed to establish stem cell lines; （2） Establishing stem cell lines from these germ cells of buffalo, both AP activity and Oct- 4 protein both are unsuitable detection markers