持续低氧对小鼠胚胎成纤维细胞增殖及饲养层制备的影响

摘要

背景:在传统的胚胎干细胞培养体系中,饲养层细胞制备和胚胎干细胞的培养扩增大都是在常氧条件下进行的,氧培养条件的改变有可能影响饲养层细胞,从而改变胚胎干细胞的生长特性或分化能力,但尚未见到这方面的系统研究报道.目的:观察低氧培养对饲养层细胞及小鼠胚胎干细胞干性维持的影响.方法:原代小鼠胚胎成纤维细胞分为常氧组(体积分数20%O2)和低氧组(体积分数5%O2)持续传代培养,绘制生长曲线,检测活性氧水平和线粒体膜电位.将小鼠胚胎干细胞分为常氧组(饲养层为常氧组小鼠胚胎成纤维细胞,体积分数20%O2条件下培养)和低氧组(饲养层为低氧组小鼠胚胎成纤维细胞,体积分数5%O2条件下培养),观察胚胎干细胞的生长形态,检测干细胞多能性指标Oct4、Sox2以及低氧诱导因子HIF-1α mRNA表达.结果与结论:①与常氧组相比,低氧组小鼠胚胎成纤维细胞增殖较快,线粒体膜电位上升,产生活性氧减少(P < 0.05);②胚胎干细胞碱性磷酸酶染色阳性,Oct4、Sox2高表达,低氧组形成的中小集落及HIF-1α mRNA表达比常氧组显著增多(P < 0.05);③结果表明,体积分数5%O2持续低氧培养利于维持饲养层细胞(小鼠胚胎成纤维细胞)的活力和胚胎干细胞的干性维持.%BACKGROUND: In traditional culture systems for embryonic stem cells, feeder cell preparation and embryonic stem cell culture are mostly performed under normoxic conditions. Changes in oxygen culture conditions are likely to influence feeder cells, thereby altering the growth characteristics or differentiation ability of embryonic stem cells, but there is still no relevant systematic report until now. OBJECTIVE: To investigate the effects of sustained hypoxia culture on the pluripotency of mouse embryonic stem cells cultured on mouse embryonic fibroblast feeder layers. METHODS: Primary mouse embryonic fibroblasts were persistently subcultured under normoxia (20% O2) and hypoxia (5% O2) conditions. Cell proliferation was measured for drawing growth curve. Reactive oxygen species level and mitochondria membrane potential of the feeder cells were detected respectively. Mouse embryonic stem cells were divided into two groups: normoxia group (plated on mouse embryonic fibroblast feeder layers under 20% O2), and hypoxia group (plated on mouse embryonic fibroblast feeder layers under 5% O2). The cell morphology was observed and the pluripotency of embryonic stem cells were detected by measurement of Oct4 and Sox2 expressions. Hypoxia inducible factor-1α mRNA expression was also tested in the four groups. RESULTS AND CONCLUSION: As compared to the normoxia group, mouse embryonic fibroblasts in the hypoxia group proliferated faster, reactive oxygen species significantly declined, and the mitochondria membrane potential level increased significantly (P < 0.05). Embryonic stem cells were positive for alkaline phosphatase, and highly expressed Oct4 and Sox2 mRNA. Much more median- or small-sized colonies formed in the hypoxia group than the normoxia group (P < 0.05). The mRNA expression of hypoxia inducible factor-1α in embryonic stem cells had a significant difference between the hypoxia and normoxia groups (P < 0.05). These findings indicate that a sustained hypoxia environment can significantly promote the viability of mouse embryonic fibroblasts as feeder layers and maintain the pluripotency of embryonic stem cells under 5% O2.