Dinitrochlorobenzene ( DNCB) was selected as the inhibitor of thioredoxin reductase ( TrxR) activi-ty to induce oxidative damage of bovine mammary epithelial cells ( BMECs) , and oxidative damage model was established by detecting cell proliferation rate, TrxR activity and antioxidant parameters in BMECs. The experi-ment was divided into two parts. The experiment 1 was conducted as a single factor randomized arrangement, and the 3th passage BMECs were divided into 35 groups with 8 replicates in each group. Cells were incubated with culture medium containing different concentrations of DNCB [ 0 ( control) , 10, 30, 50, 100, 300 and 500 μmol/L] for 2, 4, 6, 8 and 12 h to determine the appropriate action time of DNCB by detecting the cell proliferation rate. Based on the results in experiment 1, the experiment 2 was conducted as a single factor ran-domized arrangement, and the cells were divided into 7 groups with 6 replicates in each group. Cells were incu-bated with culture medium containing different concentrations of DNCB [0 (control), 100, 200, 400, 600, 800 and 1 000 μmol/L] for 2 h to select the appropriate concentration of DNCB by measuring TrxR activity and antioxidant parameters. The results showed that compared with control group, the group of 300 μmol/L DNCB for 2 h resulted in significant oxidative damage of BMECs, and cell proliferation rate reduced to 73.31%, TrxR activity decreased to 56. 23%, the activities of glutathione peroxidase, superoxide dismutase and catalase also significantly decreased ( P<0.05) , and malondialdehyde content significantly increased ( P<0.05). The results indicate that DNCB concentration of 300 μmol/L and the time of 2 h can be selected as the appropriate action concentration and time to establish the oxidative damage model of BMECs.%本试验旨在利用硫氧还蛋白还原酶( thioredoxin reductase,TrxR)的抑制剂二硝基氯苯( dinitrochlorobenzene,DNCB)作为刺激源,以细胞增殖率、TrxR活性和抗氧化指标作为判断依据,建立奶牛乳腺上皮细胞( bovine mammary epithelial cells,BMEC)的氧化损伤模型. 试验分2部分进行. 试验1采用单因子完全随机试验设计,将第3代的BMEC随机分为35组,每组8个重复. 培养液中分别添加0(对照)、10、30、50、100、300和500 μmol/L的DNCB,使之分别作用细胞2、4、6、8和12 h,通过检测细胞增殖率,初步确定适宜的作用时间;试验2在试验1得出适宜DNCB作用时间的基础上,采用单因子随机试验设计,将BMEC随机分为7组,每组6个重复,培养液中分别添加0 (对照)、10、30、50、100、300 和500 μmol/L 的 DNCB,通过检测细胞TrxR活性以及抗氧化指标,筛选出适宜的 DNCB 处理浓度. 结果表明,与对照组相比, 300 μmol/L DNCB作用2 h对BMEC产生了明显的氧化应激,细胞增殖率降到73.31%,TrxR活性降至56.23%,谷胱甘肽过氧化物酶、超氧化物歧化酶和过氧化氢酶活性也显著降低( P<0.05) ,丙二醛含量显著提高( P<0.05). 结果提示,300 μmol/L的DNCB作用浓度和2 h的作用时间可作为建立细胞氧化损伤模型的适宜剂量与作用时间.
展开▼
