硒代半胱氨酸合成蛋白基因表达对过氧化氢介导的内皮细胞EVC-304损伤的影响  

Relationship between expression of selenocysteine synthase and endothelial cell line ECV-304 injury induced by hydrogen peroxide

作　　者：赵汉东[1] 孙美娜[1] 李凤兰[1] 李晖[1] 

机构地区：[1]哈尔滨医科大学基础医学院生物化学与分子生物学教研室,150081

出　　处：《中华地方病学杂志》2014年第4期383-387,共5页Chinese Journal of Endemiology

基　　金：国家自然科学基金（30771863、81172616）

摘　　要：目的：观察硒代半胱氨酸合成蛋白（SEPSECS）基因表达对过氧化氢（H2O2）介导的氧化应激状态下内皮细胞EVC-304损伤的影响。方法体外培养人内皮细胞EVC-304，对细胞进行转染处理，分为对照组、SEPSECS低表达组、空载体转染组、SEPSECS高表达组，分别用0、200、400、600、800、1000μmol/L H2O2处理细胞6 h。转染后，用实时荧光定量PCR 法检测各组细胞SEPSECS基因表达，蛋白质免疫印迹试验（Western blot）检测各组细胞SEPSECS蛋白表达，流式细胞仪对各组细胞进行细胞周期检测。H2O2处理后，用酶联免疫吸附测定（ELISA）试剂盒检测各组细胞丙二醛（MDA）、超氧化物歧化酶（SOD）含量。结果转染后对照组、SEPSECS 低表达组、空载体转染组、SEPSECS 高表达组 EVC-304细胞 SEPSECS mRNA 表达分别为1.03±0.24、0.43±0.11、0.98±0.27、1.61±0.13，SEPSECS 蛋白表达分别为1.00±0.26、0.51±0.10、1.12±0.38、1.51±0.20。其中SEPSECS低表达组SEPSECS mRNA、蛋白表达与对照组比较，差异有统计学意义（P均〈0.05）；SEPSECS高表达组SEPSECS mRNA、蛋白表达与空载体转染组比较，差异有统计学意义（P均〈0.05）。转染后，各组细胞周期未见明显改变（P 均〉0.05）。经H2O2处理的各组EVC-304细胞MDA含量均随H2O2浓度的升高而升高。其中H2O2为800、1000μmol/L时，SEPSECS低表达组MDA含量[（15.8±0.5）、（19.6±1.5）μmol/L]明显高于对照组[（12.4±0.1）、（17.1±0.5）μmol/L，P均〈0.05]，SEPSECS高表达组MDA含量[（10.8±0.4）、（14.2±1.1）μmol/L]明显低于空载体转染组[（12.7±0.7）、（16.2±1.1）μmol/L，P均〈0.05]。经H2O2处理的各组EVC-304细胞SOD含量均随 H2O2浓度升高而降低，且 H2O2为800、1000μmol/L 时，SEPSECS 低表达组SOD 含量[（7.7±0.4）、（2.4±0.3）μmol/L]明显低于对照组[（10.0±1.0）、（6.0±0.6）μmol/L，P均〈0.05]， SEPSECS高表达组SOD含量[（1Objective To observe the influence of selenocysteine synthase（SEPSECS） on injury of human umbilical vein endothelial cell line EVC-304 induced by hydrogen peroxide （H2O2）. Methods Transfection was conducted to transfect EVC-304 which was maintained in vitro. The cells were divided into four groups： control group, SEPSECS over-expression group, empty vector group and SEPSECS silenced expression group, then Real-time PCR and Western blotting were performed to detected SEPSECS mRNA and protein expression , respectively. Flow cytometry（FCM） was performed to detect cell cycle. Different concentrations of H2O2, which including 0, 200, 400, 600, 800, 1 000 μmol/L, were used to treat EVC-304 . Then malonaldehyde （MDA）, superoxide dismutase（SOD） secreted by the cells which were treated with H2O2 for 6 h, were checked by MDA or SOD kit. Results The SEPSECS mRNA expressions of control, SEPSECS silenced expression, empty vector and SEPSECS over-expression groups were 1.03 ± 0.24, 0.43 ± 0.11, 0.98 ± 0.27 and 1.61 ± 0.13, respectively. The protein expressions of control, SEPSECS silenced expression, empty vector and SEPSECS over-expression groups were 1.00 ± 0.26, 0.51 ± 0.10, 1.12 ± 0.38 and 1.51 ± 0.20, respectively. There was a significant difference between control and SEPSECS silenced expression groups （all P〈0.05）, at the same time , this phenomenon was also observed between empty vector and SEPSECS over-expression groups （all P〈0.05）. The level of MDA was increased along with the H2O2 concentration. Besides, cell cycle was also tested, however, significant difference was not observed（all P 〉 0.05）. Meanwhile, MDA of SEPSECS silenced expression groups[（15.8 ± 0.5）,（19.6 ± 1.5）μmol/L] were significantly higher than control groups[（12.4 ± 0.1）,（17.1 ± 0.5）μmol/L, all P 〈 0.05], on the other hand, MDA of SEPSECS over-expression groups[（10.8 ± 0.4）,（14.2 ± 1.1）μmol/L] were lower than empty vector groups [（12.7 ± 0.7）,（16.2 ± 1.1）μ

关 键 词：硒代半胱氨酸合成蛋白 过氧化氢 细胞培养技术 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]