二乙烯三胺/一氧化氮聚合物诱导的奶牛乳腺上皮细胞损伤模型的建立

泌乳期间的奶牛由于代谢旺盛,往往会产生大量一氧化氮(NO),诱导发生氧化应激,进而对细胞产生毒害作用。本试验旨在探讨二乙烯三胺/一氧化氮聚合物(DETA/NO)诱导奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMEC)氧化应激的损伤条件,并建立氧化应激模型。本试验利用不同浓度(0、10、30、100、500、1 000μmol/L)的DETA/NO作为刺激源,作用BM EC 2、4、6、8、12、24 h,通过测定细胞存活率、抗氧化指标、炎症因子和NO含量及诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)活性,筛选适宜的DETA/NO作用时间及作用浓度,建立BMEC氧化损伤模型。结果表明:1 000μmol/L的DETA/NO作用细胞6 h,细胞存活率降低为74.27%,超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶活性也均显著降低(P0.05),然而,i NOS活性,NO、白细胞介素-1、白细胞介素-6、肿瘤坏死因子-α、丙二醛含量则呈现相反的变化规律,显著升高(P0.05)。综上所述,1 000μmol/L的DETA/NO处理细胞6h,可构建理想的BM EC氧化应激模型。

Establishment of Oxidative Damage Model of Bovine Mammary Epithelial Cells Induced by Diethylenetriamine/Nitric Oxide Adduct

Due to exuberant metabolism, dairy cows produce a great deal of nitric oxide ( NO) during lacta?tion, which leads to oxidative stress, and then results in cell toxicity. This experiment was conducted to investi?gate the suitable condition for oxidative damage model of bovine mammary epithelial cells ( BMEC) induced by diethylenetriamine/nitric oxide adduct ( DETA/NO) and establish an oxidative damage model. The oxida?tive damage model of BMEC was established using DETA/NO as the stimulation source. BMEC was exposed in DETA/NO with different medium concentrations (0, 10, 30, 100, 500 and 1 000μmol/L) and for differ?ent times (2, 4, 6, 8, 12 and 24 h). According to the analysis of cell survival rate and antioxidative parame?ters, inflammatory cytokines and nitric oxide ( NO) contents, as well as inducible nitric oxide synthase ( iN?OS) activity, the suitable treatment time and concentration of DETA/NO were screened. The results showed that the survival rate decreased to 74.27%, and the activitise of superoxide dismutase, catalase and glutathione peroxidase also significantly reduced ( P<0.05) after that BMEC was cultured with 1 000 μmol/L DETA/NO for 6 h. However, the activity of iNOS and the contents of NO, interleukin?1, interleukin?6, tumor necrosis factor?α and malondialdehyde showed opposite changes and increased significantly (P<0.05). In summary, the treatment of 1 000 μmol/L DETA/NO for 6 h is suitable for establishment of oxidative damage model.