过氧化氢诱导的奶牛乳腺上皮细胞氧化损伤模型的建立

金鹿; 闫素梅; 史彬林; 石惠宇; 郭晓宇; 李俊良

doi:10.3969/j.issn.1006-267x.2014.12.014

动物营养学报 >

2014 , Vol. 26 >Issue 12: 3651 - 3658

DOI: https://doi.org/10.3969/j.issn.1006-267x.2014.12.014

反刍动物营养 Ruminant Nutrition

过氧化氢诱导的奶牛乳腺上皮细胞氧化损伤模型的建立

金鹿 ,
闫素梅 ,
史彬林 ,
石惠宇 ,
郭晓宇 ,
李俊良

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内蒙古农业大学动物科学学院, 呼和浩特 010018

收稿日期: 2014-06-23

网络出版日期: 2014-12-05

基金资助

国家自然科学基金资助项目(31160466);高等学校博士学科点专项科研基金(20111515110004)

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Establishment of Oxidative Damage Model of Bovine Mammary Epithelial Cells Induced by Hydrogen Peroxide

JIN Lu ,
YAN Sumei ,
SHI Binlin ,
SHI Huiyu ,
GUO Xiaoyu ,
LI Junliang

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College of Animal Science, Inner Mongolia Agricultural University, Huhhot 010018, China

Received date: 2014-06-23

Online published: 2014-12-05

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摘要

本试验旨在利用过氧化氢(hydrogen peroxide,H₂O₂)作为刺激源,以细胞存活率及抗氧化指标为判断指标,建立奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMEC)的氧化损伤模型.试验一采用单因子完全随机试验设计,将第3代贴壁生长的BMEC随机分为42组,每组10个重复.细胞培养液中分别添加0、100、200、400、600、800和1 000 μmol/L H₂O₂,使之分别作用2、4、6、8、12、24 h,测定细胞数量,计算存活率.试验二是在试验一得出的适宜H₂O₂作用时间(本试验的结果为6 h)的基础上,采用单因子完全随机试验设计,将第3代贴壁生长的BMEC随机分为7组,每组6个重复,BMEC中添加不同浓度的H₂O₂(0、100、200、400、600、800和1 000 μmol/L)作用细胞6 h,收集细胞和培养液测定抗氧化指标,筛选使细胞发生氧化损伤的适宜H₂O₂作用浓度.试验一结果表明,600 μmol/L H₂O₂作用细胞6 h,BMEC的存活率降低至79.79%.试验二结果表明,600~1 000 μmol/L组与其他各组相比均显著降低了谷胱甘肽过氧化物酶、超氧化物歧化酶和过氧化氢酶活性,提高了丙二醛含量(P<0.05),但600、800和1 000 μmol/L组之间差异不显著(P>0.10).结果提示,H₂O₂作用浓度为600 μmol/L、作用时间为6 h,使BMEC产生了明显的氧化应激,可作为建立细胞氧化损伤模型时的适宜条件.

关键词： 奶牛乳腺上皮细胞; 过氧化氢; 氧化损伤模型

本文引用格式

金鹿 , 闫素梅 , 史彬林 , 石惠宇 , 郭晓宇 , 李俊良 . 过氧化氢诱导的奶牛乳腺上皮细胞氧化损伤模型的建立[J]. 动物营养学报, 2014 , 26(12) : 3651 -3658 . DOI: 10.3969/j.issn.1006-267x.2014.12.014

Abstract

This study was conducted to establish the oxidative damage model of bovine mammary epithelial cells (BMEC) using hydrogen peroxide (H₂O₂) as stress source and using survival rate and antioxidant parameters as judgment indices. Experiment 1 was conducted as a single factor randomized arrangement, and the 3rd passage BMEC were divided into 42 groups with 10 replicates in each group. Cells were incubated in different concentrations of H₂O₂ (0, 100, 200, 400, 600, 800 and 1 000 μmol/L,respectively) for 2, 4, 6, 8, 12 and 24 h, respectively. The cell count was determined, and the survival rate was calculated. On the basis of experiment 1 (optimal action time was 6 h), experiment 2 was also conducted as a single factor randomized arrangement, and the 3th passage BMEC were divided into 7 groups with 6 replicates in each group. Culture mediums containing different concentrations of H₂O₂ (0, 100, 200, 400, 600, 800 and 1 000 μmol/L, respectively) were used to act cell for 6 h. The cells and culture mediums were collected for the analysis of antioxidant parameters to select the appropriate concentration of H₂O₂ ensuring oxidative damage to cells. The results showed as follows: experiment 1, the BMEC survival rate reduced to 79.79% when the cells were induced by 600 μmol/L H₂O₂ for 6 h. Experiment 2, cell induced by 600 to 1 000 μmol/L H₂O₂ significantly decreased the activities of glutathione peroxidase, superoxide dismutase and catalase (P<0.05), and significantly increased malondialdehyde content compared with the other groups (P<0.05), but the differences among 600, 800 and 1 000 μmol/L groups were not significant (P>0.10). The condition that 600 μmol/L H₂O₂ reacted for 6 h is optimal for establishment of oxidative damage model in BMEC.

Key words： bovine mammary epithelial cells; hydrogen peroxide; oxidative damage model

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