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目的 :体外观察小鼠精原干细胞(spermatogonial stem cells,SSCs)的自我更新和分化能力。方法 :体外构建稳定的小鼠SSCs系,利用Ed U细胞增殖分析试剂盒检测小鼠SSCs体外自我更新的能力;利用活细胞工作站进一步观察小鼠精原干细胞体外增殖现象;运用TUNEL法检测SSCs凋亡现象;通过维甲酸(retinoic acid,RA)诱导小鼠SSCs,观察其体外分化的能力。结果:Ed U孵育小鼠SSCs 2 h后,流式检测细胞增殖率平均为40.75%;活细胞工作站下可见正在分裂的干细胞;TUNEL检测显示干细胞中凋亡信号很少;RA诱导后,标记SSCs分化的基因(C-kit、Scp3和Stra8)表达量明显升高(P<0.05)。结论:稳定构建的小鼠SSCs系与体内的SSCs类似,具有较强的自我更新以及细胞分化能力,为下一步开展精原细胞相关基因和蛋白功能研究提供了良好的技术平台。
Objective: To observe the self-renewal and differentiation of mouse spermatogonial stem cells (SSCs) in vitro. Methods: Stable mouse SSCs lines were constructed in vitro. The ability of mouse SSCs self-renewal in vitro was detected by using Ed U Cell Proliferation Assay Kit. The proliferation of mouse spermatogonial stem cells was observed by live cell workstation. The proliferation of SSCs The phenomenon of death was observed. The SSCs of mice were induced by retinoic acid (RA) and their differentiation in vitro was observed. Results: The proliferation of mouse SSCs incubated with Ed U for 2 h was 40.75% on average. Stem cells were divided under live cell workstation. TUNEL assay showed that there were few apoptotic signals in stem cells. After induced by RA, The expression of differentiated genes (C-kit, Scp3 and Stra8) was significantly increased (P <0.05). CONCLUSION: The stable mouse SSCs are similar to the SSCs in vivo and possess strong self-renewal and cell differentiation ability, which provides a good technical platform for the further research on the function of spermatogonia related genes and proteins.