中文摘要：

旨在建立牛雄性生殖干细胞（mGSCs）-支持细胞（SCs）体外共培养体系,并比较不同密度SCs共培养mGSCs的效果。分离、培养mGSCs和SCs并鉴定,然后在密度不同的SCs上共培养mGSCs,观察细胞形态、统计mGSCs集落数、测定两种细胞特异基因的相对表达量。结果表明,培养的mGSCs具有与小鼠mGSCs相同的形态特征,AKP染色细胞克隆阳性,各培养体系均表达SCs和mGSCs特异基因SCF、OCT-4;低密度SCs（225个/cm2）培养mGSCs比中密度（1 300个/cm2）和高密度（5 600个/cm2）培养mGSCs效果好：mGSCs集落纯度、体积较大;mGSCs集落比例显著（P〈0.05）和极显著增高（P〈0.01）;mGSCs特异基因表达量均极显著增高（P〈0.01）。说明建立睾丸干细胞体外共培养体系时,采用较低密度的支持细胞共培养可获得理想的睾丸生殖干细胞。

英文摘要：

This experiment was conducted to establish the co-culture system of calf mGSCs and SCs,and compare the effect of Sertoli cells（SCs）density on calf testis germ stem cells（GSCs）growth.We separated and identified mGSCs and SCs,covered GSCs on different density SCs,and then observed cell morphology,counted the colony of the GSCs and measured the relative expressions of the two cells specific genes.The results showed that the calf mGSCs have the same characteristics with mouse mGSCs,AKP strain was positive,the co-culture cells expreessed SCs′sign gene SCF and mGSCs′sign gene OCT-4;the effect of culruted mGSCs of low-density SCs（225/cm2）was better than that of medium-density SCs（1 300/cm2）and high-density SCs（5 600/cm2）：The GSC colony purity and volume were the best.The GSC colony proportion increased significantly than that in medium-density SCs cultures（P〈0.05）,increased very significantly than that in high-density SCs cultures（P〈0.01）.The qRT-PCR result showed that compared with medium-density and high-density SCs,low-density SCs made the GSC clones grow significantly well with expressions of OCT-4/SCF increased very significantly（P〈0.01）.These results indicated that the excellent testis GSCs can be acquired by low-density SCs when establishing the co-culture system of calf mGSCs and SCs.