用雄性新生仔猪生殖细胞建立干细胞系初探

Preliminary study on establishing stem cell lines from neonatal male piglet germ cells

体外培养雄性新生仔猪生殖细胞,并检测其碱性磷酸酶活性及O ct-4蛋白,探讨用雄性新生仔猪生殖细胞建立干细胞系的可行性及检测方法。结果表明,雄性新生仔猪生殖细胞在BRL细胞饲养层上培养2 d,大部分生殖细胞呈碱性磷酸酶阳性(AP+),而全部呈O ct-4蛋白阴性(O ct-4-);培养1周后,生殖细胞及细胞克隆均为AP-和O ct-4-。雄性新生仔猪生殖细胞体外培养时出现2类细胞克隆,一类体积较小,数量众多,形成1周后又开始分化,仅能传至第2代;另一类细胞克隆的形态与猪原生殖细胞来源的胚胎生殖细胞(EGC)克隆相似,细胞克隆与周围细胞分界明显,而克隆中细胞相互间界限不清,这类细胞克隆易于分化,可以传至第3代;2类细胞克隆均为AP-和O ct-4-。雄性新生仔猪生殖细胞可作为干细胞系的建系材料,AP和O ct-4蛋白均不适宜用来检测体外培养的雄性新生仔猪生殖细胞及其来源的细胞克隆。

The neonatal male piglet germ ceils were cultured in vitro and their alkaline phosphatase （AP） activity and Oct-4protein were detected in order to study the feasibility of establishing stem cell lines from them and the cell line detection methods. When neonatal male piglet germ ceils were cultured on Buffalo rat liver cell feeders for 2 days,most of them displayed AP positive and all of them showed Oct-4 protein negative,but then all showed AP and Oct 4 protein negative after a week of culture. When neonatal male piglet germ cells were cultured in vitro,two types of germ cell colonies formed. The first was smaller in size and more in quantity compared with the second. They usually began to differentiate after maintaining for about a week. The other resembled those cell colonies of EGC formed from PGCs in morphology. They were tightly packed with indistinguishable cell to cell boundaries, but had a distinct colony boundary from the surrounding feeder layers. They could be cultured up to 3 passages. Both cell colonies were AP and Oct-4 protein negative. The results of present study indicate that ： （1） neonatal male piglet germ cells can be employed to establish stem cell lines; （2） Establishing stem cell lines from these germ cells,AP activity and Oct 4 protein are unsuitable detection markers.