Bovine Embryonic Stem Cells Cultured on STO Feeder Layer Cells Treated with Domestic Mytomycin | 国产丝裂霉素处理的STO细胞作为饲养层培养牛胚胎干细胞

Chinese. 寻找一种适合牛胚胎干细胞生长的饲养层对成功培育牛胚胎干细胞有重要意义。本研究采用不同浓度国产丝裂霉素处理的SIM小鼠(Mus musculus)成纤维细胞耐硫代鸟嘌呤和耐鸟苯苷亚系(STO细胞)作为饲养层培养牛胚胎干细胞，为牛胚胎干细胞培养体系的建立提供实验依据。用10、15、20、30和40 μg/mL国产丝裂霉素分别处理1.5、3.0和4.0 h的STO细胞；形态学观察不同代次STO细胞生长状态，并对生长在以STO细胞为饲养层的牛胚胎干细胞进行碱性磷酸酶染色(AKP)、阶段特异性胚胎细胞表面抗原-4(OCT-4)和阶段特异性胚胎抗原-1(SSEA-1)免疫组化检测、体外分化形成拟胚体等实验。结果显示，当丝裂霉素的浓度为15 μg/mL处理3.0 h可有效的抑制细胞分裂；STO细胞在5~10代作为饲养层细胞形态最好且获得的牛胚胎干细胞AKP染色及OCT-4和SSEA-1抗原表达均成阳性，分别在体外分化培养形成了拟胚体。结果证明使用国产丝裂霉素能有效地处理STO细胞且在以STO细胞作为饲养层培养的牛胚胎干细胞能保持未分化状态，可以作为常规饲养层培养牛胚胎干细胞。

English. To find a suitable feeder layer for successful culture conditions of bovine embryonic stem cells is important. SIM mouse(Mus nusculus) embryo-derived thioguanine and ouabain resistant(STO) cells were treated by domestic mytomycin in different concentrations, which were used as feed layer cells to culture bovine embryonic stem cell. STO cells were treated with domestic mytomycin under 10, 15, 20, 30 and 40 μg/mL for 1.5, 3.0 and 4.0 h, respectively, and growth states of STO cells were observed in difference generations. Bovine embryonic stem cell cultured in the STO feeder layers were detected, including expression of alkaline phosphatase(AKP), octamer-binding transcription factor 4(OCT-4) and stage specific embryonic antigen 1 (SSEA-1), and formation of embryoid bodies in vitro. The results indicated that domestic mytomycin (under 15 μg/mL for 3.0 h) could inhibit effectively the division of STO cells, but not affect vitality of STO cells. Compared with conducts of Sigma, there were have the same effects, and there we not only could content the demand of preparation of feeder layers, but also could reduse the experiment costs. At the same time this research found that STO cells could be passaged proliferation in vitro long-term. However, STO cells affect the morphological structure which passed on long-term, eventually leading to the decline in the quality of feeder layers. The morphological changes of STO cells after continuous passaging indicated that of the STO cells was the better growth states than that of others generations before five generations, specially the fifth generation. Spreaded to the tenth generations, the cells occured degenerative changes and STO cells of dead rate obvious reaching to fourteenth generations. From the first to the tenth generations could been best used for feeder layers. Representative immuonofluorescence staining in the eighth passage of bovine embryonic stem cells were used. Results showed bovine embryonic stem cells cultured on STO feeder layer cells from the fifth generation to the tenth generation were in good morphology, and expressed AKP, antigen of OCT-4 and SSEA-1 in positive strongly fromed embryoid bodies. All results suggest that domestic mytomycin could inhibit effectively the division of the STO cells, and the STO cells can be used as conventional feeder layer for bovine embryonic stem cells culture.