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目的探讨低氧条件下HIF-1α和PTEN在胎鼠成纤维(MEFs)细胞增殖、分化中可能的调控作用,以及低氧对细胞周期的影响。方法低氧条件(5%O_295%N_2)下培养MEFs不同时间(0、2、4、6、8h)后,应用免疫细胞化学、RT-PCR检测方法,观察MEFs中HIF-1α和PTEN的表达并检测其细胞周期。结果免疫组化结果表明,HIF-1α及PTEN蛋白的表达均随低氧时间增长而逐渐增强。RT-PCR结果显示,HIF-1αmRNA吸光度值常氧组为(0.93±0.06),低氧2～4h组达高峰分别为(1.46±0.05)和(2.30±0.05),随后开始下降;PTENmRNA吸光度值常氧组为(0.78±0.08),低氧2～6h组逐渐达到高峰分别(1.10±0.11)和(1.61±0.23),8h开始呈现下降趋势(<0.05)。流式细胞仪分析显示,随着低氧时间延长,G0/G1期细胞从(86.72±0.41)%上升至(92.41±1.10)%,低氧使MEFs细胞周期阻滞于G1/G0期。结论低氧微环境阻滞MEFs细胞周期于G0/G1期可能与PTEN和HIF-1α过表达相关。
Objective To investigate the possible regulatory role of HIF-1α and PTEN in the proliferation and differentiation of fetal rat fibroblasts (MEFs) under hypoxic conditions and the effects of hypoxia on the cell cycle. Methods Immunocytochemistry and RT-PCR were used to detect the expression of HIF-1α and PTEN in MEFs after different time (0, 2, 4, 6, 8h) And test its cell cycle. Results The results of immunohistochemistry showed that the expression of HIF-1α and PTEN protein gradually increased with hypoxia time. The results of RT-PCR showed that the absorbance of HIF-1αmRNA was (0.93 ± 0.06) in normoxia group and (1.46 ± 0.05) and (2.30 ± 0.05) respectively in hypoxia 2 ~ 4h group, (0.78 ± 0.08) in normoxia group and 1.10 ± 0.11 (1.61 ± 0.23) in hypoxia 2 ~ 6h group (P <0.05). Flow cytometry analysis showed that the cells in G0 / G1 phase increased from (86.72 ± 0.41)% to (92.41 ± 1.10)% with hypoxia time. Hypoxia blocked MEFs cell cycle in G1 / G0 phase. Conclusion The hypoxic microenvironment block MEFs cell cycle at G0 / G1 phase may be related to PTEN and HIF-1α overexpression.