过氧化氢诱导TM3细胞氧化应激模型建立

目的构建过氧化氢(H₂O₂)诱导小鼠睾丸间质细胞(TM3)氧化应激模型, 为肥胖相关雄性生殖功能低下机制研究提供参考。方法分别以不同浓度H₂O₂处理TM3细胞4 h, 采用噻唑蓝法测定细胞活力, 酶联免疫吸附试验检测睾酮水平, 比色法测定细胞内活性氧(ROS)含量、总抗氧化能力(TAOC)、超氧化物歧化酶(SOD)活性和过氧化氢酶(CAT)活性。结果300、600 μmol/L H₂O₂组细胞存活率[(86.7±4.3)%、(46.3±2.0)%]均低于对照组[(100.0±1.7)%](P<0.05);与对照组[(250.09±26.70)pg/mL]比较, 150、300、600 μmol/L H₂O₂组TM3细胞睾酮水平[(188.93±7.06)、(179.23±6.66)、(131.59±11.26)pg/mL]均明显降低(P<0.05);与对照组比较, 150、300、600 μmol/L H₂O₂组TM3细胞SOD和CAT活性[分别为(2.42±0.17)、(2.69±0.30)、(0.44±0.41)和(56.76±12.17)、(23.45±2.17)、(24.55±18.05)U/mg protein]均明显下降(P<0.05)。结论成功构建以H₂O₂为诱导剂的TM3细胞体外氧化应激模型。

Establishment of an oxidative damage model induced by hydrogen peroxid in TM3 cells

ObjectiveTo establish an oxidative damage model with hydrogen peroxide(H₂O₂)in mouse Leydig(TM3)cells for studying possible mechanism of decreased reproductive function in obese males.MethodsAfter 4 hours' treatment with different concentration of H₂O₂, the cell viability was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)method;the testosterone level was determined with enzyme-linked immunosorbent assay(ELISA);the contents of reactive oxygen species(ROS),total antioxidant capacity(TAOC)and activities of superoxide dismutase(SOD) and catalase(CAT)were detected with spectrophotometry.ResultsThe viability of TM3 cells (86.7±4.3% and 46.3±2.0%)of 300 and 600 μmol/L H₂O₂ exposure groups decreased significantly compared to that of the control group(100±1.7%)(all P<0.05).The testosterone level(188.93±7.06,179.23±6.66,and 131.59±11.26 pg/mL)of 150,300,600 μmol/L H₂O₂ exposure groups were significantly lower than that of the control group(250.09±26.70 pg/mL)(all P<0.05).The activity of SOD(2.42±0.17,2.69±0.30,and 0.44±0.41 U/mg protein)and CAT(56.76±12.17,23.45±2.17,and 24.55±18.05 U/mg protein)of 150,300,and 600 μmol/L H₂O₂ exposure groups decreased significantly compared to those of the control group(all P<0.05).ConclusionThe TM3 cell model of oxidative injury was established successfully with the administration of H₂O₂.