体外培养成人包皮成纤维细胞周期同步化效果检测

Cell Cycle Analysis of in Vitro Cultured Human Adult Skin Fibroblasts

供体细胞和受体胞质周期协调性是核移植胚胎发育的关键,对人成体细胞的细胞周期分布及其同步化效果进行探讨,将为人治疗性克隆研究打下基础。结果发现:80%￣85%汇合生长成人包皮成纤维细胞G0/G1期细胞比例显著高于接种后第1天指数生长细胞及接种后第5天100%汇合生长细胞两组(67.0%vs58.2%,52.8%,P<0.05),指数生长状态下,仍有58.2%的成人包皮成纤维细胞处于G0/G1期;以0.2μg/ml和2μg/ml不同浓度阿菲迪霉素(aphidicolin,APD)处理成人包皮成纤维细胞24h,2μg/mlAPD组G0/G1期细胞比例显著高于0.2μg/ml组和对照组(10%FBS)(82.9%vs68.8%,63.5%,P<0.05),2μg/mlAPD可有效的将成人包皮成纤维细胞同步于G0/G1期,且培养液中添加10%、0.5%、0%FBS不同浓度血清对2μg/mlAPDG0/G1期同步化效果无显著影响(82.6%vs81.8%vs74.6%,P>0.05);以血清饥饿培养法(0.5%FBS)进行成人包皮成纤维细胞同步化处理,血清饥饿处理3d组G0/G1期细胞比例显著高于0d组(对照组)(71.8%vs59.6%,P<0.05),血清饥饿培养3d可有效将细胞同步于G0/G1期。因此,2μg/mlAPD处理24h和血清饥饿培养3d可有效将成人包皮成纤维细胞同步于G0/G1期。

The co-ordination of cell cycle between donor cells and recipient cytoplasm was the key aspect of reconstructed embryos development, studies on cell cycle characteristics of cells at different proliferation stages and the effects of synchronization of cell cycle were the foundation of therapeutic cloning research, As results, we found that the rates of G0/G1 cells at 80%-85% confluency stage were significant higher than the cells at exponential growth stage and 100% confluency stage （67.0% vs 58.2%, 52.8%, P〈0.05）, 58.2% of human adult skin fibroblasts reached the G0/G1 stage even cells at exponential growth stage; 2 μg/ml aphidicolin （APD） treated group showed better results for synchronization of G0/G1 phases than 0.2 μg/ml and 0 μg/ml group （82.9% vs 68.8%, 63.5%, P〈0.05）. Furthermore, there was no significant difference in synchronization of G0/G1 phases along with 2 μg/ml APD combined with 0%, 0.5%, and 10% FBS respectively （74.6% vs 81.8% vs 82.6%, P〉0.05）, the synchronization of cell cycle mainly monitored by 2 μg/ml APD when different concentration of serum coexist; Synchronization of cell cycle could effectively achieved by serum deprivation for 3 d, which was significant higher than control groups （71.8% vs 59.6%, P〈0.05）. Thus it could be concluded that 2 μg/ml APD treated for 24 h and serum deprivation for 3 d could synchronize the cell cycle effectively.