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目的探讨硫酸镍(NiSO4)致原代培养大鼠睾丸间质细胞DNA氧化损伤的作用。方法选择健康成年雄性Wistar大鼠,采用胶原酶消化以及Percoll分离液梯度密度离心法,经贴壁培养获得高纯度大鼠睾丸间质细胞,采用3β-HSD法鉴定细胞纯度。取对数生长期的间质细胞,分别用浓度为0、250、500和1000μmol/L硫酸镍处理6、12和24 h。采用流式细胞仪检测细胞内活性氧(ROS)水平;分光光度法检测间质细胞抑制羟自由基能力(Anti-OH·),酶联免疫吸附法(ELISA)检测细胞内8-羟基脱氧鸟苷(8-OHdG)含量。结果经3β-HSD法鉴定,睾丸间质细胞纯度达95%以上。与对照组比较,250μmol/L硫酸镍处理12 h组和500、1000μmol/L硫酸镍各处理组,ROS水平均增高(P<0.05);250μmol/L硫酸镍处理12和24 h组,以及500、1000μmol/L硫酸镍各处理组,Anti-OH·均降低(P<0.05);500、1000μmol/L硫酸镍处理12和24 h组,8-OHdG含量均增加(P<0.05)。结论硫酸镍致大鼠睾丸间质细胞DNA氧化损伤可能与其导致间质细胞Anti-OH·降低和ROS水平增加有关。
Objective To investigate the effect of nickel sulfate (NiSO4) on DNA oxidative damage of primary cultured rat Leydig cells. Methods Healthy adult male Wistar rats were used to digest collagenase and Percoll gradient centrifugation. The purity of testicular stromal cells was determined by 3β-HSD. Logarithmic growth phase stromal cells were treated with 0, 250, 500 and 1000 μmol / L nickel sulfate for 6, 12 and 24 h, respectively. The level of reactive oxygen species (ROS) in the cells was detected by flow cytometry. Anti-OH · was measured by spectrophotometry and the levels of 8-hydroxydeoxygenated oxygen in the cells were detected by enzyme-linked immunosorbent assay (ELISA) Glycoside (8-OHdG) content. Results By 3β-HSD method, the purity of testicular stromal cells was more than 95%. Compared with the control group, the levels of ROS increased (P <0.05) at 250μmol / L nickel sulphate for 12 h and 500, 1000μmol / L nickel sulphate treatment groups; (P <0.05). The contents of 8-OHdG in 500 and 1000μmol / L nickel sulfate treated groups increased (P <0.05) at 12 and 24 h. Conclusion The DNA oxidative damage induced by nickel sulfate in rat testicular stromal cells may be related to the reduction of stromal cells and the increase of ROS level.