猪原始生殖细胞的分离、培养与鉴定  

Isolation, culture and characterization of porcine primordial germ cells

作　　者：李世杰[1] 郑瑞珍[2] 岳奎忠[1] 陈瑛[1] 杨增明[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]中国科学院遗传与发育生物学研究所,北京100080

出　　处：《动物学报》2003年第4期529-533,共5页ACTA ZOOLOGICA SINICA

基　　金：国家自然科学基金资助项目 (No .3 973 0 2 5 0 )~~

摘　　要：Embryonic germ cells (EG cells) are pluripotential undifferentiated stem cells isolated from cultured primordial germ cells (PGCs). Like ES cells, EG cells are of importance for gene targeting, therapeutical cloning and organ transplantation. The aim of this study was to isolate and characterize EG cells from porcine PGCs. The genital ridges from 24-26 days old porcine embryos were treated in 0 02% EDTA for 20 min and pricked with a needle to release PGCs. The isolated PGCs were cultured on a SNL feeder layer in an EG cell medium. The EG cell medium consisted of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% Buffalo rat lever (BRL) cell-conditioned medium, 15% fetal bovine serum, 1 mmol L-glutamine, 0 1 mol nonessential amino acids, 10 μmol β-mercaptoethanol and antibiotics. The freshly isolated PGCs were positive for alkaline phosphatase activity and Periodic acid-Schiff’s staining. Under this culture regime, PGCs could be maintained in an undifferentiated state and used for further cultures. One strain of the cultured PGCs was cultured 8 times, and alkaline phosphatase activity was detected in the colony formed from this strain. These cultured PGCs could spontaneously differentiate into fibroblast-like cells. These data suggested that we had successfully isolated EG-like cells from porcine PGCs .Embryonic germ cells (EG cells) are pluripotential undifferentiated stem cells isolated from cultured primordial germ cells (PGCs). Like ES cells, EG cells are of importance for gene targeting, therapeutical cloning and organ transplantation. The aim of this study was to isolate and characterize EG cells from porcine PGCs. The genital ridges from 24-26 days old porcine embryos were treated in 0 02% EDTA for 20 min and pricked with a needle to release PGCs. The isolated PGCs were cultured on a SNL feeder layer in an EG cell medium. The EG cell medium consisted of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% Buffalo rat lever (BRL) cell-conditioned medium, 15% fetal bovine serum, 1 mmol L-glutamine, 0 1 mol nonessential amino acids, 10 μmol β-mercaptoethanol and antibiotics. The freshly isolated PGCs were positive for alkaline phosphatase activity and Periodic acid-Schiff’s staining. Under this culture regime, PGCs could be maintained in an undifferentiated state and used for further cultures. One strain of the cultured PGCs was cultured 8 times, and alkaline phosphatase activity was detected in the colony formed from this strain. These cultured PGCs could spontaneously differentiate into fibroblast-like cells. These data suggested that we had successfully isolated EG-like cells from porcine PGCs .

关 键 词：猪 原始生殖细胞 分离 培养 鉴定 胚胎干细胞 

分 类 号：Q813.1[生物学—生物工程]