过氧化氢诱导斜带石斑鱼原代肝细胞氧化损伤模型的构建

本试验以斜带石斑鱼原代培养肝细胞为研究对象,以过氧化氢为刺激源,以肝细胞存活率和抗氧化指标的变化为判断指标,旨在建立稳定的斜带石斑鱼原代肝细胞氧化损伤模型。在原代肝细胞培养液中分别添加0(对照)、100、200、400、600、800和1 000μmol/L过氧化氢,使之分别作用2、4、6、8、12和24 h,共42组,每组10个重复,测定肝细胞存活率。在得出适宜过氧化氢作用时间的基础上,使每个浓度的过氧化氢(每个过氧化氢浓度设6个重复)作用于肝细胞适宜时间后,收集肝细胞和培养液测定抗氧化指标,筛选使肝细胞发生氧化损伤的适宜过氧化氢作用浓度。结果显示:800μmol/L过氧化氢作用肝细胞8 h,斜带石斑鱼肝细胞的存活率降低至61.98%;800和1 000μmol/L组与其他各组相比,肝细胞超氧化物歧化酶、谷胱甘肽过氧化物酶(600μmol/L组除外)和过氧化氢酶活性显著降低(P0.05),丙二醛与脂质过氧化物含量显著升高(P0.05),但800和1 000μmol/L组之间差异不显著(P0.05)。以上结果表明,过氧化氢作用浓度为800μmol/L、作用时间为8 h,可作为建立斜带石斑鱼肝细胞氧化损伤模型的适宜条件。

Establishment of Oxidative Damage Model of Primary Hepatocytes of Grouper ( Epinephelus coioides) Induced by Hydrogen Peroxide

This experiment was aimed to establish the stable oxidative damage model of primary hepatocytes of grouper using primary cultured hepatocytes of grouper ( Epinephelus coioides) as study object, hydrogen per-oxide (H2O2) as stress source and the changes of hepatocytes survival rate and antioxidant indexes as judgment indexes. The concentrations of 0 (control), 100, 200, 400, 600, 800 and 1000 μmol/L H2O2 were added in the cultured fluid of primary hepatocytes, and were cultured by 2, 4, 6, 8, 12 and 24 h, respectively. There were 42 groups and each group had 10 replicates. After culturing, the survival rate of hepatocytes was detected. Based on the appropriate action time was obtained, each concentration of H2O2(each concentration of H2O2 had 6 replicates) was used to culture hepatocytes for appropriate time. After culturing, the hepatocytes and cultured fluid were collected to determined antioxidant indexes, in order to select the appropriate action concentration of H2O2 ensuring oxidative damage to hepatocytes. The results showed that the survival rate of hepatocytes induced by 800μmol/L H2O2 at 8 h was reduced to 61.98%. The experiment also revealed that the 800 and 1000 μmol/L group was significantly decreased the activities of superoxide dismutase, glutathione peroxidase (except 600 μmol/L group) and catalase (P<0.05), and significantly increased the contents of malondialdehyde and lipid peroxidation compared with other groups ( P<0. 05 ) . However, the 800 and 1000 μmol/L groups had no significant differences (P>0.05). The results manifest that 800 μmol/L H2O2 incubated for 8 h can be used as a suitable method to establish oxidative damage model of primary hepatocytes of grouper.