钒诱导蛋鸡输卵管膨大部上皮细胞氧化应激模型的建立

发布时间：2018-06-28 05:45

本文选题：钒 + OMECs　；参考：《畜牧兽医学报》2017年02期

【摘要】：本试验旨在通过蛋鸡输卵管膨大部上皮细胞(OMECs)的原代培养,构建利用偏钒酸铵(V5+)建立氧化应激模型的方法。试验选用开产前2~3周龄健康罗曼蛋鸡,分离、培养及鉴定OMECs。利用单因素设计6个不同水平的钒浓度(0、25、50、100、250、1 000μmol·L~(-1))处理12h后,测定细胞活力、细胞凋亡、细胞活性氧(ROS)的生成,细胞内超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的活性,丙二醛(MDA)的含量,细胞上清液中乳酸脱氢酶(LDH)的释放量。结果表明:1)分离培养的细胞24h后形成细胞岛,呈"铺路石样";免疫荧光染色细胞抗细胞角蛋白(CK18)、卵清蛋白(OVA)、雌激素受体1(ESR1)和孕酮受体(PGR)呈阳性,抗波形蛋白呈阴性。2)50和100μmol·L~(-1)的钒处理OMECs 12h后,细胞活力分别下降到(69.90±2.78)%和(63.60±1.57)%,显著低于25μmol·L~(-1)钒处理组(P0.05),但是显著高于250和1 000μmol·L~(-1)钒处理组(P0.05)。3)细胞凋亡率随着钒添加量的增多而升高,且各处理组差异显著(P0.05);1 000μmol·L~(-1)的钒细胞凋亡率为最高(P0.05)。4)ROS的测定结果表明,100和250μmol·L~(-1)钒处理组的FITC相对平均值显著高于0μmol·L~(-1)钒处理组(P0.05),且250μmol·L~(-1)钒处理组的FITC相对平均值显著高于100μmol·L~(-1)钒处理组(P0.05)。5)50μmol·L~(-1)钒处理组SOD活性显著低于0μmol·L~(-1)钒处理组(P0.05),但MDA含量差异不显著。而100μmol·L~(-1)钒处理组的MDA含量显著升高(P0.05);50和100μmol·L~(-1)钒处理组细胞CAT活性显著低于0μmol·L~(-1)钒处理组(P0.05);LDH释放量随着钒的添加剂量的增加而升高,1 000μmol·L~(-1)钒处理组LDH的释放量最高,且100μmol·L~(-1)钒处理组LDH的释放量显著高于0μmol·L~(-1)钒处理组(P0.05),达到141.61±2.81U·gprot-1。综上表明,OMECs原代细胞培养成功,在OMEC原代细胞里添加100μmol·L~(-1)的V5+处理12h,可建立OMECs氧化应激模型。
[Abstract]:The purpose of this study was to establish an oxidative stress model using ammonium metavanadate (V5) through primary culture of oviduct expanded epithelial cells (OMECs) of laying hens. OMECs were isolated, cultured and identified from healthy Roman laying hens at the age of 2 and 3 weeks before birth. Six different levels of vanadium were designed by single factor design. After 12 h treatment, cell viability, cell apoptosis, production of reactive oxygen species (Ros), activities of superoxide dismutase (SOD) and catalase (cat), and malondialdehyde (MDA) content were measured. The release of lactate dehydrogenase (LDH) in cell supernatant. The results showed that the cells isolated and cultured 24 hours later were "paving stone like", immunofluorescence staining showed positive expression of anti-cytokeratin (CK18), ovalbumin (OVA), estrogen receptor 1 (ESR1) and progesterone receptor (PGR), and positive expression of cytokeratin (CK18), ovalbumin (OVA), estrogen receptor 1 (ESR1) and progesterone receptor (PGR). The cell viability of OMECs decreased to (69.90 卤2.78)% and (63.60 卤1.57) after 12 h treatment with vanadium of 50 and 100 渭 mol L ~ (-1), respectively, which was significantly lower than that of 25 渭 mol L ~ (-1) vanadium treatment (P0.05), but significantly higher than that of 250 and 1 000 渭 mol L ~ (-1) vanadium treatment (P0.05). The apoptosis rate of vanadium cells was the highest (P0.05) in the vanadium cells treated with 渭 mol L-1 and 250 渭 mol L-1. The results showed that the relative average value of FITC in the vanadium treated group was significantly higher than that in the 0 渭 mol L-1 group (P0.05), and the relative average value of FITC in the 250 渭 mol L-1 vanadium treatment group was significantly higher than that in the vanadium treatment group (P0.05), and the relative average value of FITC in the vanadium treated group was significantly higher than that in the vanadium treatment group (P0.05). Sod activity in 50 渭 mol L ~ (-1) vanadium treatment group was significantly lower than that in 0 渭 mol L ~ (-1) vanadium treatment group (P0.05), but there was no significant difference in MDA content. The activity of cat in mol 渭 mol L ~ (-1) vanadium treated group was significantly lower than that in 0 渭 mol L ~ (-1) vanadium treatment group (P0.05), and the release of LDH was the highest in 1 000 渭 mol L ~ (-1) vanadium treatment group with the increase of vanadium additive content, but the content of MDA in the vanadium treatment group was significantly higher than that in the 0 渭 mol L ~ (-1) vanadium treatment group (P < 0.05), and the content of LDH in the vanadium treated group was significantly lower than that in the vanadium treatment group (P < 0.05). The release of LDH in the 141.61 渭 mol L ~ (-1) vanadium treatment group was significantly higher than that in the 0 渭 mol L ~ (-1) vanadium treatment group (P0.05), and reached 141.61 卤2.81 U / g prot-1. The results showed that the primary cells of OMECs were cultured successfully, and the oxidative stress model of OMECs could be established by adding 100 渭 mol L-1 V5 to the primary cells for 12 h.
【作者单位】：四川农业大学动物营养研究所动物抗病营养教育部重点实验室抗病营养与饲料农业部重点实验室;
【基金】：国家自然科学基金(青年项目31402031) 四川省教育厅(青年基金13ZB0290) 科技部科技支撑计划(2014BAD13B04) 四川省科技厅(2014NZ0043;2014NZ0002;2013NZ0054)
【分类号】：S831

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