摘要
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500μmol/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500μmol/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,300μmol/L DNCB作用2 h对BM EC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,300μmol/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。
Dinitrochlorobenzene（ DNCB） was selected as the inhibitor of thioredoxin reductase（ TrxR） activity 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 experiment was divided into two parts. The experiment 1 was conducted as a single factor randomized arrangement,and the 3 th 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 and500 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 randomized arrangement,and the cells were divided into 7 groups with 6 replicates in each group. Cells were incubated 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 300μmol /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.
出处
《动物营养学报》
CAS
CSCD
北大核心
2015年第12期3984-3990,共7页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金资助项目(31160466)
"十二五"农村领域国家科技计划课题(2012BAD12B09-03)
关键词
奶牛乳腺上皮细胞
二硝基氯苯
氧化损伤模型
bovine mammary epithelial cells
dinitrochlorobenzene
oxidative damage model
