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[目的]探究诺考达唑和阿非科林联合应用对人胚胎干细胞周期同步化到G1和S期的作用并观察药物对细胞生长和生物功能的影响。[方法]采用诺考达唑和阿非科林先后作用于胚胎干细胞,流式细胞仪测定细胞周期,显微镜下观察药物对细胞生长状态的影响,荧光染色技术观察胚层标志物α-SMA和β-tubulin表达。[结果]加入诺考达唑和阿非科林后,G1期为66.35%,S期为33.52%,多数细胞被同步化在G1期;诺考达唑作用16 h,G1期为18.45%,S期为79.10%,多数细胞从周期抑制中恢复;能够形成典型的拟胚体;荧光染色显示α-SMA和β-tubulin均有表达。[结论]诺考达唑和阿非科林联合作用能够使胚胎干细胞66.35%同步化在G1期,诺考达唑作用16 h,79.10%的细胞同步化在S期。中胚层标志物α-平滑肌肌动蛋白和外胚层标志物β微管蛋白在蛋白水平均有明显表达。去除药物后,细胞未发生显著性损伤。
[Objective] To explore the effect of nocodazole and aphidicolin on the synchronization of human embryonic stem cell cycle to G1 and S phases and to observe the effect of drugs on cell growth and biological function. [Methods] The effects of nocodazole and aphidicolin on embryonic stem cells were studied. The cell cycle was determined by flow cytometry. The effects of drugs on cell growth were observed under microscope. The germ layer markers α-SMA and β -tubulin expression. [Results] After nocodazole and alfaconil were added, the G1 phase was 66.35% and the S phase was 33.52%. Most of the cells were synchronized in G1 phase. The effect of nocodazole was 16 h and the G1 phase was 18.45% S phase was 79.10%. Most of the cells recovered from the cycle inhibition. The typical embryoid bodies were formed. Fluorescence staining showed that both α-SMA and β-tubulin were expressed. [Conclusion] The combination of nocodazole and alfaconter could synchronize 66.35% embryonic stem cells in G1 phase, 16 h Nocodazole, 79.10% cells in S phase. Mesoderm markers α-smooth muscle actin and ectoderm markers β tubulin in the protein levels were significantly expressed. After removing the drug, the cells did not show significant damage.