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目的 筛选合适的保持人表皮干细胞特性的体外培养基,并观察其生物学性状。 方法 按不同培养基分组,配制FAD培养基和FAD加入不同浓度的碱性成纤维细胞生长因子(basic fibroblast growth factor,b FGF)的培养基,即FAD、FAD1、FAD2、FAD3等组,以及角质化细胞专用无血清培养基(keratinocyte- SFM,K- SFM) ,同时应用同一个体的成纤维细胞经丝裂霉素处理后作滋养层,培养人表皮干细胞。通过细胞生长曲线及MTT检测,观察细胞的生长状态。在体外培养扩增表皮干细胞后,利用透射电镜、流式细胞仪分析、Brd U检测,观察细胞的生长状态;结合流式细胞仪分析,观察细胞周期的变化。 结果 人表皮干细胞在FAD组中生长良好,加入b FGF可进一步改善细胞的生长状态。扩增后细胞检测提示,人表皮干细胞在体外培养过程中呈典型的克隆性生长,生长周期长。流式细胞仪检测表明,人表皮干细胞80 .2 %处于G0 /G1 期;透射电镜观察见其细胞器少,细胞核浆比大。 结论 加入b FGF的FAD培养基并同时利用滋养层,更适合人表皮干细胞的生长,在稳定状态下是一种处于静止期的幼稚细胞。
Objective To screen suitable culture media of human epidermal stem cells and observe its biological characteristics. Methods FAD, FAD1, FAD2 and FAD3 groups were prepared by adding different concentrations of basic fibroblast growth factor (FGF) into FAD medium and FAD group according to different media. Human keratinocyte-SFM (K-SFM) and human fibroblasts were treated with mitomycin C and used as trophoblast to culture human epidermal stem cells. Through the cell growth curve and MTT assay, observe the cell growth status. After culturing and expanding epidermal stem cells in vitro, the cell growth was observed by transmission electron microscopy, flow cytometry and BrdU detection. The changes of cell cycle were observed by flow cytometry. Results Human epidermal stem cells grew well in the FAD group. The addition of b FGF could further improve the cell growth status. Expansion of the cell test showed that human epidermal stem cells cultured in vitro showed a typical clonal growth, long growth cycle. Flow cytometry showed that 80.2% of human epidermal stem cells were in G0 / G1 phase. The number of organelles and the ratio of nucleus to cytoplasm were large under transmission electron microscope. Conclusion Addition of bFGF to FAD medium and the simultaneous use of trophoblast are more suitable for the growth of human epidermal stem cells, which are a kind of naive cells in quiescent state.