不同影响因素对水牛原始生殖细胞分离培养的影响

Influence of different factor on the isolation and culture of the buffalo PGCs

探讨了不同影响因素（性别、胎龄、分离方法）对水牛原始生殖细胞（PGCs）分离培养的影响。结果显示：PGCs多层堆积生长形成克隆,该克隆具有明显的边界与饲养层区分;PGCs表达AKP和OCT-4;公胎牛组的克隆总数和克隆直径都显著高于母胎牛组,（179和32,（227.76±83.84）μm和（102.34±40.59）μm）,P〈0.05;在原代克隆数目上,80～90 d组只有16个克隆,显著低于其他各组（P〈0.05）,在克隆直径上,80～90 d组为（190.63±65.11）μm,也显著低于除70～80 d组外的其他各组（P〈0.05）,并且在最高传代数上,80～90 d组仅为3代,而其他各组都达到或超过了6代;机械分离组在克隆总数和直径上都显著高于胰酶消化组,（186和30,（216.95±77.41）μm和（140±38.76）μm）,P〈0.05,并且机械分离组的最高传代数为8代,而酶消化组仅为3代。结果表明,本试验分离培养出的水牛PGCs具有与已经分离培养出来的其他物种PGCs相似的生物学特性;公胎牛、胎龄小于80 d以及使用机械分离方法更利于水牛PGCs的分离培养。

The influence of different factor （sex, fetal age and dissociating ways） on the isolation and culture of the buffalo PGCs were investigated in this study. The cells formed large,multilayered colonies with distinct boundaries. PGCs expressed AKP and OCT-4. The male fetus group was significantly higher than female fetus group in the PGCs clones number and diameter （179 vs 32,（227.76±83.84）/,m vs （102.34±40.59）μm,P〈0.05）. In the number of colonies,the 80-90 d group was just 16 colonies, which was significantly （P〈0.05） lower than other groups. And in the diameter of colonies,the 80-90 d group was（190.63±65, 11）μm,whichwas also significantly （P〈0. 05） lower than other groups except for the 70 80 d group. In the maximal passage,the 80-90 d group was just three passages,but other groups were more than six passages. And there were significantly （P〈0.05） different between me- chanical and trypsin method in the number and diameter of EG-like cell colonies （186 vs 30, （216.95±77.41）/,m vs （140±38.76）μm）. In the maximal passage,the mechanical group was eight passages,but trypsin group were just three passages. In conclusion,buffalo PGCs had morphology and characterization similar to those of the established PGCs in other animal, male fetus,fetus younger than 80 d and mechanical dissociating method facilitate to the isolation and culture of buffalo PGCs.