犊牛睾丸生殖干细胞与支持细胞体外共培养体系的建立

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（1300个／cm2）和高密度（5600个／cm2）培养 mG-SCs 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）；mG-SCs 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。%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 separa-ted and identified mGSCs and SCs,covered GSCs on different density SCs,and then observed cell morphol-ogy,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 cul-ruted 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 pro-portion increased significantly than that in medium-density SCs cultures (P <0.05),increased very signifi-cantly 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 excel-lent testis GSCs can be acquired by low-density SCs when establishing the co-culture system of calf mGSCs and SCs.