连二亚硫酸钠诱导大鼠肝细胞氧化应激模型的建立  

Oxidative Stress Model of BRL-3A Cells Induced by Sodium Hydrosulfite

作　　者：孙晨 戈胜 宁夏青 王佳琦 张暖暖 王晓然 张士霞[1] SUN Chen;GE Sheng;NING Xiaqing;WANG Jiaqi;ZHANG Nuannuan;WANG Xiaoran;ZHANG Shixia(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071000,China;Yidu Animal Husbandry Service Center of Hubei Province,Yichang 443300,China)

机构地区：[1]河北农业大学动物医学院,保定071000 [2]湖北省宜都市畜牧服务中心,宜昌443300

出　　处：《中国畜牧兽医》2023年第6期2217-2223,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31802250);河北省教育厅科学技术研究项目(BJ2019053)。

摘　　要：【目的】探讨连二亚硫酸钠(Na_(2)S_(2)O_(4))诱导大鼠肝细胞(BRL-3A)氧化应激模型的制备方法,以期建立一种稳定的BRL-3A细胞氧化应激模型。【方法】利用含Na_(2)S_(2)O_(4)的培养基造成细胞缺氧,更换正常培养基对BRL-3A细胞进行复氧培养,造成细胞氧化应激损伤。将BRL-3A细胞分为5组,空白对照组(0 mmol/L Na_(2)S_(2)O_(4))在细胞培养箱中正常培养,试验组给予含不同浓度(0.1、1、5、10 mmol/L)Na_(2)S_(2)O_(4)的培养基进行缺氧培养4 h、复氧培养2 h的处理后,采用CCK-8法检测细胞存活率。采用荧光探针DCFH-DA法检测细胞中活性氧(ROS)的释放,利用试剂盒检测细胞培养液中乳酸脱氢酶(LDH)活性,以及BRL-3A细胞中丙二醛(MDA)、谷胱甘肽(GSH)含量及超氧化物歧化酶(SOD)活性。【结果】BRL-3A细胞经过缺氧/复氧处理后,与空白对照组相比,随着Na_(2)S_(2)O_(4)浓度的增加,试验组BRL-3A细胞存活率逐渐下降(P<0.01),ROS释放量逐渐增高(P<0.01),细胞培养液中LDH活性及细胞内MDA含量呈浓度依赖性增高(P<0.01),细胞内GSH含量及SOD活性呈浓度依赖性降低(P<0.05;P<0.01)。【结论】利用含5 mmol/L Na_(2)S_(2)O_(4)的培养基对BRL-3A细胞进行缺氧培养4 h、复氧培养2 h的处理后,可以建立稳定的BRL-3A细胞氧化应激模型。【Objective】This study was aimed to explore the preparation method of rat hepatocyte(BRL-3A)oxidative stress model induced by sodium hydrosulfite(Na_(2)S_(2)O_(4)),in order to establish a stable oxidative stress model of BRL-3A cells.【Method】The medium containing Na_(2)S_(2)O_(4) was used to cause hypoxia of cells,and the normal medium was replaced to reoxygenate BRL-3A cells,thus causing oxidative stress damage of cells.BRL-3A cells were divided into 5 groups,and the control group was cultured normally in a cell incubator(0 mmol/L Na_(2)S_(2)O_(4)),the experiment group was treated with different concentrations(0.1,1,5 and 10 mmol/L)of Na_(2)S_(2)O_(4) for 4 h of hypoxia and 2 h of reoxygenation,then the cell viabiliaty rate was detected by CCK-8 assay.The fluorescent probe DCFH-DA method was used to detect the release of reactive oxygen species(ROS)in cells.The activity of lactate dehydrogenase(LDH)in cell culture medium,the contents of malondialdehyde(MDA)and glutathione(GSH),and the activity of superoxide dismutase(SOD)in BRL-3A cells were detected by kit.【Result】After hypoxia/reoxygenation treament,compared with blank control group,with the increase of Na_(2)S_(2)O_(4) concentration,the viability rate of BRL-3A cells was gradually decreased(P<0.01),the release of ROS was gradually increased(P<0.01),the activity of LDH and the content of MDA were increased in a concentration dependent manner(P<0.01),the content of GSH and the activity of SOD were decreased in a concentration dependent manner(P<0.05 or P<0.01).【Conclusion】This study showed that a stable oxidative stress model of BRL-3A cells could be established which was induced by 5 mmol/L Na_(2)S_(2)O_(4) for hypoxia of 4 h and reoxygenation of 2 h.

关 键 词：BRL-3A细胞 连二亚硫酸钠(Na_(2)S_(2)O_(4)) 氧化应激 

分 类 号：S852.21[农业科学—基础兽医学]