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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。
Objective To investigate the effect of hypoxic treatment on differentiated cardiomyocytes during different stages of embryonic stem cell differentiation. Methods The experimental method of inducing differentiation of embryonic stem cells into cardiomyocytes was established. Hypoxia (4%) and hypoxia (about 20%) were used as controls at different stages of differentiation. Differentiation was identified by immunofluorescence After cardiomyocytes, flow cytometry was used to detect the effects of hypoxia on the proliferation, differentiation and apoptosis of differentiated cardiomyocytes. Results The differentiated cells were detected by immunofluorescence. Some of the cells were positive for α-actinin (red) and positive for cardiac troponin I (green). The flow cytometry showed that compared with normoxia group, The ratio of α-actinin positive cells and cTnI positive cells increased by 12.55% (P <0.01) and 6.11% (P <0.05), respectively, and the apoptosis rate in hypoxia group was significantly higher than that in normoxia group (P < 0.01). The ratio of cells in S phase in hanging drop hypoxia group was significantly lower than that in normoxia group (P <0.01). Conclusion During the process of directional differentiation of embryonic stem cells into cardiomyocytes, the suspension-phase hypoxia treatment increased the ratio of myocardial specific protein positive cells, the rate of apoptosis was stable, the cell proliferation ability decreased due to the maturation of cells, The best solution for hypoxia treatment.