培养时间和传代次数对山羊输卵管上皮细胞周期和凋亡的影响

Effect of days after confluence and passages on the cell cycle and apoptosis of goat oviductal epithelial cells

目的利用山羊输卵管上皮细胞（OECs）探讨体外培养时间和传代次数对细胞生长的影响。方法建立山羊OECs的体外培养体系,用流式细胞术分别对半数贴壁、刚贴满（贴壁1d）、贴满3d和贴满5d,以及原代、Ⅲ代和Ⅴ代细胞分别进行细胞周期和凋亡检测。结果细胞贴满3d和传至Ⅲ代时,细胞凋亡比率较低,处于静止期（G0/G1）细胞明显降低,极性线粒体受损明显降低;培养时间延长和传代次数增加后,凋亡比率显著升高,处于增殖期（S,G2/M）的比率显著降低,极性线粒体受损明显升高。结论采用OECs培养胚胎时,细胞应控制在Ⅲ代以内,且贴满时间不超过3d。

Objective The experiment was conducted with the objective of determining effects of culture time and subculture on the growth of goat oviductal epithelial cells （OECs）. Methods We firstly succeeded in culturing goat OECs in vitro,and then the cell cycle was examined by flow cytometric analysis and cell apoptosis was determined by Annexin-V and PI staining in goat OECs at different days of confluence and different passages at day 1 of confluence. ／Results／The percentages of （G0/G1） stage,late apoptotic ＋ necrotic and cells with reduced number of polarized mitochondria decreased when OECs were subcultured within Ⅲ passage and confluent less than three days. However,the percentages of S ＋ G2/M stage decreased but late apoptotic ＋ necrotic and cells with reduced number of polarized mitochondria increased with prolonged culture and passage. Conclusion A better development of co-cultured embryos was achieved with somatic cells within Ⅲ passage and less than three days after confluence.