氢气对子前期胎盘缺血/再灌注细胞模型氧化应激水平的调控作用

Regulative effect of hydrogen on oxidative stress level of placental ischemia-reperfusion cell model of preeclampsia

目的探讨氢气在子前期胎盘缺血/再灌注细胞模型中的细胞保护作用及可能机制。方法体外培养的人绒毛膜癌细胞株JAR分为空白组、常氧培养条件下含饱和氢气组(常氧+H2组)、缺血/再灌注模型组(模型组)、缺血/再灌注饱和氢气干预组(模型+H2组)和缺血/再灌注维生素C阳性对照组(模型+VC组)。模型组、模型+H2组和模型+VC组。细胞在缺氧(5%CO2、95%N2)环境下无糖无血清培养2 h后,更换完全培养基,在常氧(5%CO2、95%空气)环境下培养,建立缺血/再灌注细胞模型,在缺氧及复氧期分别给予氢气或维生素C等干预。各组细胞在完全培养基中培养24 h后,采用MTT法检测细胞存活率,化学比色法检测细胞内丙二醛(MDA)含量及超氧化物歧化酶(SOD)的活性,Real-Time PCR检测SOD mRNA的表达;各组细胞在完全培养基培养1 h后,采用荧光探针2',7'-二氢二氯荧光素二乙酸酯(DCFH-DA)检测细胞内活性氧类(ROS)水平。结果与模型组比较,模型+H2组细胞存活率升高(P<0.05),ROS产生及MDA含量明显减少(P<0.01,P<0.05),SOD活性增强(P<0.01),SOD mRNA表达水平升高(P<0.05)。结论氢气对子前期胎盘缺血/再灌注细胞模型有细胞保护作用,其机制可能是通过上调SOD-mRNA的转录、增强SOD酶活性及降低细胞内ROS水平,从而降低细胞氧化应激水平。

Objective To investigate the effects of hydrogen on cell protection of placental ischemia-reperfusion cell model of preeclampsia and its possible mechanism. Methods JAR cens cultured in vitro were divided into five groups, i.e. the blank control group, saturated hydrogen group under normal oxygen cultivation conditions （ normal oxygen ＋ H2 group）, ischemia-reperfusion model group （ model group）, ischemia-reperfusion group with saturated hydrogen intervention （model ＋H2 group）, and ischemia-reperfusion positive control group with vitamin C intervention （model ＋ VC group）. Cells of the model group, model ＋ H2 group, and model ＋ VC group were cultured with sugar and serum free medium in hypoxia （5% CO2 and 95% N2） environment for 2 h, and then they were cultured with high sugar and 100/0 serum medium in the environment of 95% air and 5% CO2. The ischemia-reperfusion cell model was established. The hydrogen intervention and vitamin C intervention were performed during the hypoxia period and reoxygenation period, respectively. After cells were cultured in the complete medium for 24 h, the cell survival rate was detected by the MTT method; the content of malondialdehyde （MDA） and the activity of superoxide dismutase （SOD） were measured by the chemical colorimetry; and the expression of SOD mRNA was detected by the Real-Time PCR. After cells were cultured in the complete medium for 1 h, the intracellular ROS level was measured by the DCHF-DA fluorescence. Results Compared to the model group, the cell survival rate of the model ＋ H2 group increased （P〈0.05） ; the ROS generation （P〈0.01） and MDA content （P〈0.05） decreased significantly; and the SOD activity （P〈 0.01） and the expression level of SOD mRNA （P 〈 0.05） increased. Conclusion Hydrogen has the protective effect on the placental ischemia-reperfusion cell model of preeclampsia. The mechanism may be the reduction of cell oxidative stress level by up-regulating the transcription of SOD-mRNA, increasing the activity of SOD, and decreasing the level of intracellular reactive oxygen.