山羊乳腺上皮细胞氧化损伤模型的建立

doi:10.16431/j.cnki.1671-7236.2022.05.032

摘要/Abstract

摘要： 【目的】探究过氧化氢(H₂O₂)诱导山羊乳腺上皮细胞(GMEC)氧化损伤的最佳条件，构建可靠、稳定的氧化损伤模型，为今后探究紫大薯花青素对氧化损伤的保护机制提供模型条件。【方法】试验采用二因子完全随机设计，第一因素为H₂O₂处理浓度，分别为0(对照组)、350、430、460、490、520和550 μmol/L，第二因素为对GMEC的处理时间，分别为8、11、14、17 h。舍弃细胞存活率低于60%的处理组，并根据结果重新调整处理浓度与时间。然后通过细胞内谷胱甘肽过氧化物酶(GSH-Px)的活性、活性氧(ROS)和丙二醛(MDA)的含量以及培养液内乳酸脱氢酶(LDH)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)的活性和总抗氧化能力(T-AOC)的测定确定最佳的H₂O₂处理条件。【结果】与对照组相比，各H₂O₂处理组作用8 h后GMEC存活率均显著降低(P<0.05)，其中350、430、460 μmol/L H₂O₂处理8 h时细胞存活率分别降低至64.6%、72.9%、66.2%，基本符合细胞存活率的筛选需求(70%)。因此，选择350、430、460、490、520 μmol/L H₂O₂处理4、6、8、10 h进行后续试验。调整后，除4 h 430 μmol/L处理组外，各处理组LDH活性在不同处理时间下均显著高于对照组(P<0.05)，并随着时间的延长逐渐升高，10 h时各处理组LDH活性均达到最大值，且与浓度呈正比;各处理组ROS含量在6 h时均显著高于对照组(P<0.05)，且8 h时各处理组ROS含量较6 h均大幅度显著升高(P<0.05)，说明8 h为ROS含量变化的转折点;430、490、520 μmol/L处理组MDA含量在处理8 h后均显著高于对照组(P<0.05)，其中430 μmol/L处理组MDA含量在8和10 h时显著高于对照组(P<0.05);各浓度组CAT、SOD、GSH-Px和T-AOC活性在处理6~8 h时均出现不同程度的降低，且8 h时仅430 μmol/L处理组CAT、SOD、GSH-Px和T-AOC活性显著低于照组(P<0.05)。【结论】 430 μmol/L H₂O₂处理GMEC 8 h为构建GMEC氧化损伤模型最佳条件。

关键词: 过氧化氢; 山羊乳腺上皮细胞; 氧化损伤; 存活率; 抗氧化

Abstract: 【Objective】 This experiment was conducted to investigate the optimal conditions of oxidative damage induced by H₂O₂ in goat mammary epithelial cell (GMEC),and construct a reliable and stable oxidative damage model,in order to provide model conditions for exploring the protective mechanism of anthocyanins on oxidative damage in Dioscorea alata L.【Method】 In this experiment,two factors complete random design was used,the first factor was the concentration of H₂O₂ (0 (control group),350,430,460,490,520 and 550 μmol/L),and the second factor was the treatment time of GMEC (8,11,14 and 17 h),respectively.Treatment groups with cell survival rate lower than 60% were discarded,and treatment concentration and time were readjusted according to the results.Subsequently,the activity of glutathione peroxidase (GSH-Px),the content of reactive oxygen species (ROS) and malondialdehyde (MDA) in cells,the activity of lactate dehydrogenase (LDH),catalase (CAT),superoxide dismutase (SOD) and the total antioxidant capacity (T-AOC) in culture medium were measured by the kit,so as to determine the best treatment mode of H₂O₂.【Result】 Compared with control group,the survival rate of GMEC in all treatment groups were significantly decreased after 8 h treatment (P<0.05).The survival rate of GMEC in 360,430 and 460 μmol/L treatment groups were reduced to 64.6%,72.9% and 66.2% after 8 h treatment,which basically met the screening requirement of cell survival rate (70%).Therefore,350,430,460,490 and 520 μmol/L H₂O₂ treatments were selected for 4,6,8,10 h for follow-up tests.After adjustment, the LDH activity in all treatment groups was significantly higher than that of control group at different treatment times (P<0.05), and gradually increased with the extension of time, except 430 μmol/L treatment group for 4 h. The LDH activity of all treatment groups reached the maximum value at 10 h, and was proportional to the concentration. Compared with control group, ROS content in all treatment groups at 6 h was significantly increased (P<0.05), and ROS content in all treatment groups at 8 h was significantly increased compared with 6 h (P<0.05), indicating that 8 h was the turning point of ROS content change. Compared with control group, MDA content in 430, 490 and 520 μmol/L treatment groups was significantly increased after 8 h treatment (P<0.05), and MDA content in 430 μmol/L treatment group was higher than that of control group (P<0.05). The activities of CAT, SOD, GSH-Px and T-AOC in all groups were decreased to varying degrees from 6 to 8 h after treatment, and only the activities of CAT, SOD, GSH-Px and T-AOC in 430 μmol/L treatment group were significantly lower than that of control group at 8 h (P<0.05).【Conclusion】 Therefore,the method that GMEC treated with 430 μmol/L H₂O₂ for 8 h met the need of constructing oxidative damage model.

Key words: hydrogen peroxide; goat mammary epithelial cells; oxidative damage; survival rate; antioxidant

中图分类号:

S858.27

张元锌, 王闫宇, 云艳虹, 陈俊朴, 石惠宇, 王学梅. 山羊乳腺上皮细胞氧化损伤模型的建立[J]. 中国畜牧兽医, 2022, 49(5): 1917-1925.

ZHANG Yuanxin, WANG Yanyu, YUN Yanhong, CHEN Junpu, SHI Huiyu, WANG Xuemei. Establishment of Oxidative Damage Model of Goat Mammary Epithelial Cells[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1917-1925.

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