PDF(Pubmed)
Abstract：
The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.
摘要：
美洲狮的数量在不断减少,通过体细胞核移植克隆可用于保存物种。决定克隆胚胎发育成功的因素之一是供体细胞的细胞周期阶段。我们评估了完全汇合的效果(~100%)，血清饥饿（0.5%血清），和roscovitine（15µM）处理对Puma皮肤来源的成纤维细胞的G0/G1细胞周期同步的流式细胞术分析。此外，我们评估了这些同步方法对形态学的影响，生存能力，和使用显微镜工具的细胞凋亡水平。结果表明，培养24h的细胞汇合(84.0%)，48小时（84.6%），72h（84.2％）和血清饥饿96h（85.4％）产生的停滞在G0/G1期（P0.05）的细胞百分比明显高于未进行任何细胞周期同步方法的细胞（73.9％）。然而，而血清饥饿降低了活细胞的百分比，完全汇合和roscovitine治疗无差异（P0.05）。此外，roscovitine12h(78.6%)和24h(82.1%)不能同步G0/G1期细胞(P0.05)。总之,完全汇合在G0/G1阶段诱导美洲狮成纤维细胞细胞周期同步，而不影响细胞活力。这些结果对于计划供体细胞在美洲狮中进行体细胞核转移可能是有价值的。
