中国组织工程研究2018，Vol.22Issue(9)：1450-1456,7.DOI:10.3969/j.issn.2095-4344.0478
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
Effects of sustalned hypoxia on proliferation of mouse embryonic fibroblasts and preparation of feeder layers
摘要
Abstract
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.关键词
小鼠胚胎成纤维细胞/小鼠胚胎干细胞/饲养层/低氧/线粒体膜电位/活性氧/低氧诱导因子/干性/干细胞/国家自然科学基金分类
医药卫生引用本文复制引用
魏含清,裴轶劲,王丹丹,蒋杨,王春,李洪梅..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[J].中国组织工程研究,2018,22(9):1450-1456,7.基金项目
国家自然科学基金项目(81173136) （81173136）
广东省科技计划项目(2013B022000003) （2013B022000003）
广东省基础与应用基础研究专项(广东省自然科学基金)(2015A030313524) （广东省自然科学基金）
广东省东莞市国际科技合作项目(2015508102004) （2015508102004）
广东医科大学科研基金自然科学类面上项目(M2015019) （M2015019）
the National Natural Science Foundation of China,No.81173136 （）
the Scientific Research Project of Guangdong Province,No.2013B022000003 （）
the Natural Science Foundation of Guangdong Province,No.2015A030313524 （）
the International Scientific Cooperation Project of Dongguan in Guangdong Province,No.2015508102004 （）
the Scientific Research Foundation of Guangdong Medical University,No.M2015019 （）
