低氧对小鼠胚胎干细胞诱导分化成心肌细胞的影响

目的探讨在胚胎干细胞诱导分化过程的不同阶段进行低氧处理对分化心肌细胞的影响。方法建立胚胎干细胞向心肌细胞诱导分化的实验方法,在分化过程的不同阶段采取低氧（氧浓度为4%）处理,并设立常氧组（约为20%）作为对照,用免疫荧光鉴定分化后的心肌细胞,以流式细胞术检测低氧对分化心肌细胞的增殖、分化、凋亡的影响。结果分化细胞经免疫荧光检测显示,部分细胞呈α辅肌动蛋白阳性（红色）、心肌肌钙蛋白I阳性（绿色）;分化细胞经流式检测显示,与常氧组相比,悬滴低氧组α-actinin阳性细胞和cTnI阳性细胞的比率分别提高了12.55%（P〈0.01）和6.11%（P〈0.05）,悬浮低氧组凋亡比率与常氧组相比明显提高（P〈0.01）,悬滴低氧组处于S期的细胞比率与常氧组相比明显降低（P〈0.01）。结论在胚胎干细胞向心肌细胞的定向诱导分化过程中,采用悬滴阶段低氧处理,使心肌特异性蛋白阳性细胞比率提高,细胞凋亡率稳定,细胞增殖能力因细胞分化成熟而有所降低,为低氧处理的最佳方案。

Effects of hypoxia on mouse embryonic stem cells differentiation into cardiomyocytes

Objective To explore the effects of hypoxia on embryonic stem cells differentiation into cardiomyocytes at different differentiation stages.Method The way of inducing differentiation experiments of embryonic stem cells into cardiomyocytes was established.At the different differentiation stages,hypoxia（oxygen concentration is 4%）as experimental group,normal oxygen（oxygen concentration is 20%） as normoxic group,differentiated cardiomyocytes were identified by immunofluorescence and the effects of hypoxia on cell proliferation,differentiation,and apoptosis of differentiated cardiomyocytes were measured.Results Immunofluorescence staining showed that part of differentiated cardiomyocytes were α-actinin positive（red）and cardiac troponin I positive（green）.Compared with normoxic group,flow cytometry results showed that α-actinin positive cells increased by 12.55%（P 0.01） and cTnI positive cells increased by 6.11%（P 0.05） in the hanging-drop group of low oxygen.Compared with normoxic group,the proportion of apoptotic cells significantly increased（P 0.01） in the suspension-hypoxia group.Compared with normoxic group,the proportion of cells in S phase significantly decreased（P 0.01） in the hanging-drop-hypoxia group.Conclusion In the phase of amplification and hanging-drop,hypoxia enhances cardiac differentiation of embryonic stem cells,and the hanging-drop-hypoxia group is the best;While in the suspension-hypoxia period,hypoxia is not conducive to the differentiation.