小鼠精原干细胞体外培养体系的建立

Establishment of a Long-Term Culture System for Mouse Spermatogonial Stem Cells in vitro

目的：建立小鼠精原干细胞体外扩增的培养体系。方法：取出生后2～6d的雄性ICR小鼠30只，脱颈法处死，切取睾丸后，采用两步酶消化法制成单细胞悬液，添加特定培养液，以小鼠胚胎成纤维细胞作为饲养层细胞进行体外培养。培养后通过BrdU整合实验检测其增殖能力，并用碱性磷酸酶（AP）染色、免疫荧光及RT-PCR技术对培养细胞进行鉴定。结果：小鼠精原干细胞在体外稳定增殖，所得克隆AP强阳性，标记物检测GFRα-1^＋／Oct-4^＋／VASA^＋／SCP3^-，基因表达GFRα-1^＋／Oct-4^＋／SCP3^-，证实所培养细胞为处于未分化状态的小鼠精原干细胞。结论：在本研究建立的培养体系下小鼠精原干细胞可稳定传代并保持未分化状态，为探索体外精子发生过程提供了良好的研究平台。

Objective： To establish a long-term proliferation culture system for mouse spermatogonial stem cells. Methods： Testis tissues were obtained from 30 newborn male ICR mice on postnatal day 2-6. Testis cell suspension was collected by two-step enzymatic digestion prior to culture. The dissociated cells were aliquoted into tissue culture plates and cultivated with a modified system composed of serum-free defined medium on mouse embryonic fibroblasts （MEF） feeders. Their proliferation was determined by the BrdU incorporation test and the cultured cells identified by alkaline phosphatase （AP） activity, immunofluorescence staining and RT-PCR assay. Results： The cultures remained in a steady state and continued to generate germ cell colonies. The undifferentiated state was confirmed by strong positivity for AP activity, immunofluorescent staining of GFRα-1^＋/Oct-4^＋/VASA^＋/SCP3^ - and GFRα-1^＋/Oct-4^＋/SCP3^ - at the gene expression levels. Conclusion： Mouse spermatogonial stem cells could be expanded in our defined culture system and passaged steadily in vitro. The harvested cells remained in an undifferentiated state, which has provided a good platform for the study of spermatogenesis in vitro.