过氧化氢介导人瓣膜间质细胞产生氧化应激细胞模型的建立

Establishment of oxidative stress model with human valve interstitial cells mediated by H_2O_2

目的建立过氧化氢(hydrogen peroxide,H2O2)介导的瓣膜间质细胞(valve interstitial cells,VICs)氧化应激模型,为心脏瓣膜病始发机制的研究以及未来抗氧化治疗药物筛选提供细胞学模型。方法将分离培养的原代人主动脉瓣VICs随机分为对照组和处理组,分别以含10%胎牛血清的DMEM培养液和普通培养液+不同浓度梯度(0、50、100、300、500、800、1 000μmol/L)的H2O2进行培养。采用H-E染色、MTT比色法、AnnexinⅤ/PI双染流式细胞术检测细胞生存能力和凋亡的发生。结果处理24h后,MTT结果显示各组VICs细胞存活率差异有统计学意义(P<0.01),H2O2浓度在50、100μmol/L时细胞存活率与对照组相比升高(P<0.05),随后细胞存活率随H2O2浓度升高开始下降,在800μmol/L时出现快速降低的拐点,此时细胞存活率为(69.8±8.3)%,1 000μmol/L时细胞存活率降为(14.3±11.0)%。H-E染色显示在800μmol/L时VICs形态皱缩,胞核固缩。流式细胞检测则进一步证实800μmol/L时VICs出现明显凋亡,此时多为中晚期凋亡。结论 H2O2最佳作用浓度为800μmol/L,作用时间为24h,在该条件下可成功建立氧化应激细胞模型。

Objective To establish an oxidative stress model of human valve interstitial cells （VICs） mediated by hydrogen peroxide （H2O2）, so as to provide cytology model for research on pathogenesis of valvular heart diseases and screening anti-oxidant drugs. Methods The isolated and cultivated primary human VICs were divided into different groups randomly.. control group containing DMEM culture medium with 10% FBS, experimental groups were treated with different concentrations （0, 50, 100, 300, 500, 800, and 1000 μmol/L） of H2O2. H-E staining was used to observe cell morphology, MTT assay was used to estimate cell viability, and Annexin V/PI flow cytometer was employed to evaluate the apoptosis of VICs. Results MTT assay showed that the survival rates of VICs were significantly different 24 h after exposure to different concentrations of H2O2 （P〈0. 01）, with those in 50 and 100μmol/L groups being significantly higher than that of control group （P〈0.05）, and the cell survival rate began to decrease with the increase of H202 concentrations; the decrease became quicker when H202 concentrations was 800 μmot/L, with a survival rate of （69.8±8.3） % ; and the survival rate decreased to （14.3±11.0） % when the concentration reached 1 000 μmol/L. H-E staining showed that at 800 μmol/L, H202 resulted in crimple and karyopyknosis of the VICs. Flow cytometer results confirmed apparent apoptosis of VICs at the concentration of 800 μmol/L, with the apoptosis already in the middle and advanced stages. Conclusion Oxidative stress model of VICs can be successfully established with H2O2, with the optimum concentration of H2O2 being 800 μmol/L and the expose period being 24 h.