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目的探讨低氧条件下小鼠皮肤成纤维细胞内低氧调节关键转录因子HIF-1、多潜能转录因子Oct4及表观遗传调控因子Uhrf1的表达,为低氧微环境影响细胞重编程效率的机制研究奠定基础。方法将体外培养的小鼠皮肤成纤维细胞分为低氧(2%O_2+5%CO_2+93%N_2)6h、12h、24h、48h组和常氧(20%O_2+5%CO_2)对照组。用免疫荧光化学方法和RT-PCR检测小鼠皮肤成纤维细胞中HIF-1,Oct4和Uhrf1的表达,在蛋白和m RNA水平对其进行分析。结果成纤维细胞内HIF-1、Oct4、Uhrf1蛋白的阳性表达率随着低氧时间的延长逐渐增多,常氧组均未见表达。低氧6h、12h、24h组m RNA表达量均比常氧组高,但差异不显著(>0.05);而低氧48h组三个基因m RNA表达明显高于常氧组(<0.01)。结论低氧微环境HIF-1,Oct4和Uhrf1在成纤维细胞内表达水平上调,提示它们在其中发挥了重要作用。
Objective To investigate the hypoxia-induced hypoxia-regulated key transcription factor HIF-1, pluripotency transcription factor Oct4 and epigenetic regulator Uhrf1 in hypoxia-induced hypoxia microenvironment, Research laid the foundation. Methods The mouse skin fibroblasts cultured in vitro were divided into two groups: hypoxia (2% O2 + 5% CO2 + 93% N2) for 6h, 12h, 24h, 48h and normoxic (20% O2 + 5% CO2) . Immunofluorescence staining and RT-PCR were used to detect the expression of HIF-1, Oct4 and Uhrf1 in mouse skin fibroblasts, and their protein and mRNA levels were analyzed. Results The positive rates of HIF-1, Oct4 and Uhrf1 in fibroblasts gradually increased with the prolongation of hypoxia time, and no expression was found in normoxia group. The expression of m RNA in hypoxia 6h, 12h and 24h groups was higher than that in normoxia group, but the difference was not significant (> 0.05). The m RNA expression in hypoxia 48h group was significantly higher than that in normoxic group (<0.01). Conclusion Hypoxia microenvironment HIF-1, Oct4 and Uhrf1 in fibroblasts upregulated, suggesting that they play an important role in them.