| 摘要: |
| 【目的】研究高非酯化脂肪酸（NEFA）对体外培养奶牛肝细胞的氧化应激状态、氧化抗氧化酶活性及mRNA表达的影响。【方法】分离奶牛肝细胞，培养72 h后，添加不同浓度（0.0，0.3，0.6，1.2和2.4 mmol/L）的NEFA继续培养12，24和36 h，采用酶联免疫吸附试验（ELISA）法,检测过氧化氢（H2O2）、丙二醛（MDA）含量及总抗氧化能力（TAC）、羟自由基抑制能力和超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化氢酶（CAT）的活性，实时荧光定量PCR（qRT-PCR）法检测GSH-Px、SOD、CAT基因mRNA的相对表达量。【结果】随着NEFA浓度的增加，添加NEFA 12，24和36 h时，各处理组肝细胞中H2O2和MDA含量增加，TAC含量、SOD、GSH Px、CAT的活性以及羟自由基抑制能力降低。与对照组相比，添加NEFA 12和36 h时，SOD mRNA相对表达量均随NEFA浓度的增加呈剂量依赖性降低；添加NEFA 24 h时，0.6，1.2和2.4 mmol/L NEFA处理组的SOD mRNA相对表达量均极显著降低（P＜0.01）。添加NEFA 12 h时，各浓度NEFA处理组GSH-Px mRNA 相对表达量均极显著降低（P＜0.01）；添加NEFA 24 h时，1.2和2.4 mmol/L NEFA处理组的GSH-Px mRNA 相对表达量均极显著降低（P＜0.01），0.6 mmol/L NEFA处理组的GSH-Px mRNA相对表达量显著降低（P＜0.05）；添加NEFA 36 h时，GSH-Px mRNA相对表达量随NEFA浓度的增加呈剂量依赖性降低。添加NEFA 12 h时，0.6和2.4 mmol/L NEFA处理组的CAT mRNA相对表达量均极显著降低（P＜0.01）；添加NEFA 24和36 h时，CAT mRNA相对表达量呈剂量依赖性降低。【结论】NEFA诱导体外培养的奶牛肝细胞发生氧化还原失衡，尤其以高浓度NEFA诱导的氧化应激更为严重。 |
| 关键词: 非酯化脂肪酸 奶牛 肝细胞 氧化应激 |
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| 基金项目:国家自然科学基金项目（31502136）;安徽省自然科学基金项目（1608085QC56）;安徽农业大学青年科学基金重点项目（2014zr008）;安徽农业大学稳定和引进人才科研项目（yj2015-14）;安徽省现代农业产业技术体系项目（2016-2020） |
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| Effect of NEFA on redox state of hepatocytes in vitro from dairy cow |
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DONG Jihong,,WU Jinjie,LIN Genxiang,et al
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| Abstract: |
| 【Objective】This study investigated the effect of non-esterified fatty acids (NEFA) on oxidative stress state,activity and mRNA expression of antioxidant enzymes of dairy cow hepatocytes in vitro.【Method】Dairy cows hepatocytes were stimulated for another 12,24 and 36 h by NEFA at different concentrations （0.0,0.3,0.6,1.2,and 2.4 mmol/L) after 72 h culture since separation.The contents of H2O2,MDA and TAC,inhibition of hydroxyl radical capacity,and activities of SOD,GSH-Px and CAT were measured by enzyme-linked immunosorbent assay (ELISA).The mRNA expressions of GSH-Px,SOD and CAT were measured by real-time quantitative PCR (qRT-PCR). 【Result】Contents of H2O2 and MDA increased with the increase of NEFA concentration,while TAC,activities of SOD,GSH Px,and CAT as well as the inhibition of hydroxyl radical capacity decreased.Compared with the control group,mRNA relative expression of SOD had a dose-dependent decrease at 12 and 36 h and reduced (P＜0.01) very significantly at 24 h in 0.6,1.2,and 2.4 mmol/L NEFA groups.The mRNA relative expression of GSH-Px reduced (P＜0.01) very significantly at 12 h in all groups,reduced (P＜0.01) very significantly at 24 h in 1.2 and 2.4 mmol/L NEFA groups,and reduced (P＜0.05) significantly in 0.6 mmol/L NEFA group.It had dose dependently reduced effect at 36 h.The mRNA relative expression of CAT reduced (P＜0.01) very significantly at 12 h in 0.6 and 2.4 mmol/L NEFA groups,and had dose dependently reduced effect at 24 and 36 h. 【Conclusion】NEFA can induce the redox imbalance in primary culture hepatocytes in vitro,especially at high concentrations. |
| Key words: non-esterified fatty acids dairy cow hepatocytes oxidative stress |
