缺氧诱导因子1α对缺氧条件下大鼠心肌细胞糖酵解的影响

An experimental study on the influence of hypoxia induction factor-1α on the glycolysis of the rat myocardial cell under hypoxic condition

目的观察缺氧诱导因子1α(HIF-1α)对缺氧状态下大鼠心肌细胞糖酵解的影响。方法常规分离、培养大鼠心肌细胞,分为单纯缺氧组:使用无糖培养基在低氧混合气体(体积分数94%N2、5%CO2、1%O2)中培养;HIF-1α抑制组:先利用RNA干扰技术构建HIF-1α蛋白低表达的细胞模型,再作上述缺氧培养。两组细胞均设缺氧前(常规培养)及缺氧1、3、6、12、24h6个时相点,采用生物化学方法检测细胞中糖酵解关键酶己糖激酶(HK)、磷酸果糖激酶(PFK)、乳酸脱氢酶(LDH)的活性以及培养上清液中乳酸(LA)的含量。结果(1)HK、PFK活性:与缺氧前比较,两组心肌细胞两酶活性均呈先升高后下降的变化趋势。单纯缺氧组两酶活性峰值分别为(159±13)、(298±44)U/g.HIF-1α抑制组两酶活性在缺氧1、3、6h时均明显低于单纯缺氧组(P<0.05或0.01),其峰值分别为(133±55)、(188±55)U/g.(2)LDH活性、LA含量:与缺氧前(92±12)U/g比较,单纯缺氧组细胞LDH活性缺氧后显著升高,6h时达高峰(2568±125)U/g(P<0.01),随后逐渐下降;各时相点培养上清液中LA含量也成倍增加。HIF-1α抑制组细胞的LDH活性于缺氧3h时达峰值(2125±126)U/g,明显高于缺氧前(P<0.01);培养上清液中LA含量亦较缺氧前增高(P<0.01),但增幅较小。结论缺氧条件下大鼠心肌细胞中HIF-1α高表达是细胞糖酵解持续增强的直接原因,此为心肌细胞应对缺氧环境的重要内源性保护机制。

Objective To investigate the influence of hypoxia induction factor-1α（HIF-1α） on glycosis of rat myocardial cell under hypoxic condition. Methods The myocardial cells of the rats were routinely isolated and cultured. The cells were divided into single hypoxia （H） and HIF-1α inhibiting （I） groups. The cells in H group were cultured in glucose-free medium with mixed low-oxygen gas [ 1%O2, 94% N2 and 5% CO2 （v/v） ]. While the cells in I group were cultured with low-oxygen gas after the cell model of low expression of HIF-1α protein constructed by RNAi technique. The cells in both groups were all observed before hypoxia （routine culture） and at the time points of 1, 3, 6, 12 and 24 hours of hypoxia. The LA （lactate acid ） content in the supernatant of the culture and the activity of the key enzymes in glycolysis such as hexokinase （HK） , phosphofructokinase （PFK） and lactate dehydrogenase （LDH） of both groups of cells were determined at all the time points. Results （ 1 ） After hypoxia, the HK and PFK activities of the rat myocardial cells in H and I groups were obviously increased at the beginning and decreased thereafter when compared with that before hypoxia. While the activities of HK and PFK in H group at 1,3 and 6 hours after hypoxia were evidently higher than those in I group（ P 〈0.05 or 0.01 ） , and the peak activity of them in H and I groups was 159 ± 13 U/g vs 133 ± 55 U/g ,and 298 ±44 U/g vs 188 ± 55 U/g, respectively. （2） Compared with normal control（92 ± 12 U/g） , the LDH activity of the cells in H group after hypoxia increased significantly, reaching the peak at 6 hours after hypoxia（2 568 ± 125 U/g, P 〈0.01 ） , and it decreased thereafter, while that in I group peaked at 3 hours after hypoxia（2 125 ± 126 U/g, P 〈0.01 ） . The LA content in the culture supernatant in H group increased significantly after hypoxia with the passage of time, while that in I group increased in smaller magnitude （ P 〈 0.01 ）. Conclusion High expression of HIF-1α in the rat myocardial cells after hypoxia could directly cause continuous enhancement of cell glycolysis, which was beneficial to the protection of myocardial cells under hypoxic condition.