过氧化氢诱导人胎盘滋养细胞HTR-8/SVneo氧化应激模型的建立

Establishment of hydrogen peroxide-induced oxidative stress model in HTR-8/SVneo cells

目的通过研究不同浓度过氧化氢(H2O2)作用不同时间对人胎盘滋养细胞HTR-8/SVneo氧化应激水平的影响,确定建立滋养细胞氧化应激模型的条件。方法将HTR-8/SVneo用不同浓度(125、250、500μmol/L)H2O2分别处理不同时间(1、2、4、24h)后,采用CCK-8法检测细胞存活率;流式细胞术分析检测细胞内超氧化物阴离子水平以及细胞凋亡情况;紫外分光光度法检测细胞培养上清液中超氧化物歧化酶(superoxide dismutase,SOD)活性以及乳酸脱氢酶(lactate dehydrogenase,LDH)含量。结果随H2O2浓度增加及作用时间延长,HTR-8/SVneo细胞存活率逐渐下降,细胞内超氧化物阴离子含量及细胞凋亡率不断增加,细胞培养上清液中SOD活性逐渐降低、LDH含量不断升高。其中在250μmol/L H2O2作用4h条件下,SOD活性明显低于对照组(P<0.05),细胞内超氧化物阴离子水平及细胞凋亡率较对照组均显著增加(P<0.05),但LDH漏出量无明显增多(P>0.05),且该条件下细胞有较高的存活率(85.13%)。结论建立人胎盘滋养细胞HTR-8/SVneo氧化应激模型的最适条件为250μmol/L H2O2作用4h。

Objective To establish the oxidative stress model of human trophoblast cells by studying the effects of hydrogen peroxide （H2O2） on the cell line HTR-8/SVneo. Methods HTR-8/SVneo cells were treated with different concentrations （125,250,500 umol/L） of H2 02 for different time （1, 2,4,24 hours）. Cell viability was measured by CCK-8 assay. Superoxide dismutase （SOD） activity and lactate dehydrogenase （LDH） leakage in cell culture supernatant were detected by ultraviolet spectrophotometry. Intracellular superoxide anion and apoptosis proportion were measured by flow cytometry. Results Following the increasing of H202 concentration and the lengthening of the action time, cell viability and SOD activity decreased. Meanwhile, LDH leakage, apoptosis proportion andintracellular superoxide anion production increased. When treated with 250 umol/L H202 for 4 hours, SOD activity significantly decreased （P〈0.05）, apoptosis proportion and intracellular superoxide anion production significantly increased compared with the control group （P〈0.05）. However, the increase of LDH leakage was not that significant （P〉0.05）, and cell viability could reach to 85.13% in the above-mentioned condition. Conclusions The most appropriate condition of establishing the oxidative stress model of HTR-8/SVneo is treatment with 250 /zmol/L H2 02 for 4 hours.