彗星电泳法和K-SDS法检测甲醛和H_2O_2对人脐静脉内皮细胞DNA的损伤

DNA Damage of Human Umbilical Vein Endothelial Cells Caused by Formaldehyde and H_2O_2 with Comet Assay and K-SDS Assay

背景与目的:研究甲醛、H2O2以及两者共同作用对人脐静脉内皮细胞DNA的损伤。材料与方法:①应用彗星电泳法于荧光显微镜下观察人脐静脉内皮细胞经甲醛、H2O2、甲醛和H2O2不同浓度(0、5、10、25、50、100 μmol/L)作用20 min或25 μmol/L浓度作用不同时间(0、10、20、30 min)后的DNA损伤情况;②用K-SDS法检测甲醛、甲醛和H2O2不同浓度(0、10、50、100、500、1 000、2 000 μmol／L)或50 μmol／L、100 μmol／L浓度不同时间(0、0.5、1、1.5、2、4 h)引起的DNA-蛋白质的交联效应。结果:①彗星电泳结果:内皮细胞与不同浓度甲醛孵育20 min,5、10、25 μmol／L甲醛所致DNA损伤以断裂为主且与阴性对照有统计学意义(P<0.05);与等摩尔H2O2共同孵育时,5、10、25μmol／L浓度组均可使单独H2O2作用时的彗星尾距增加,50、100 μmol／L使单独H2O2作用时的彗星尾距减小;25μmol／L甲醛作用不同时间,尾距随时间增加而增大(P<0.05)。②交联结果:内皮细胞与不同浓度甲醛孵育1.5 h后,引起DNA-蛋白质交联(DPC)形成明显增高(P<0.05);与等摩尔H2O2共同孵育时除1 h组(P<0.05),其余各组与阴性对照无统计学差异;50 μmol／l的甲醛作用不同时间,2 h组与阴性对照有统计意义(P<0.05);100 μmol／L的甲醛、甲醛与H2O2作用不同时间,各?

BACKGROUND & AIM: To investigate DNA damage of human umbilical vein endothelial cells after treated with formaldehyde, H2O2, formaldehyde and H2O2. MATERIAL AND METHODS: ①Comet assay was employed to assess DNA damage of human umbilical vein endothelial cells treated with either various concentrations (0, 5, 10, 25, 50, 100μmol/L) of formaldehyde, H2O2, formaldehyde and H2O2 for 20 min or 25μmol/L formaldehyde, H2O2, formaldehyde and H2O2 for different time(0, 10, 20, 30 min) to quantify the DNA damage. ②K-SDS was employed to evaluate DNA-Protein cross-link(DPC) of endothelial cells treated with either various concentrations of formaldehyde, formaldehyde and H202(0, 10, 50, 100, 500, 1 000, 2 000 μmol/L) for 1.5 h or 50 μmol/L and 100 mol/L formaldehyde, formaldehyde and H2O2 for different time(0,0.5,1,1. 5,2,4 h) to quantify the DPC. RESULTS: DNA breakage caused by 5, 10, 25μmol/L formaldehyde was significantly different from control, and might be increased by H2O2. Tail moment was time-dependent when treated with 25μmol/L formaldehyde. The formation of DPC increased(P < 0.05)after treated with various concentrations of formaldehyde for 1.5 h. CONCLUSION:①Formaldehyde can cause DNA breakage(< 25μmol/L) and DPC( > 500 μmol/L), both were concentration and time-dependent.②H2O2 can cause DNA breakage, which was concentration and time-dependent.