钒诱导蛋鸡输卵管膨大部上皮细胞氧化应激模型的建立

doi:10.11843/j.issn.0366-6964.2017.02.018

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (2): 340-350.doi: 10.11843/j.issn.0366-6964.2017.02.018

钒诱导蛋鸡输卵管膨大部上皮细胞氧化应激模型的建立

黄选洋,王建萍^*,丁雪梅,白世平,曾秋凤,张克英

(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，成都 611130)

收稿日期:2016-07-29 出版日期:2017-02-23 发布日期:2017-02-23
通讯作者: 王建萍，副研究员，博士，主要从事禽的营养研究，E-mail:wangjianping1983@hotmail.com
作者简介:黄选洋（1991-），男，四川广汉人，硕士生，主要从事禽的营养研究，E-mail：hxy1819@sina.com
基金资助:
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）

Establishment of an Oxidative Stress Model Induced by Vanadium in Oviduct Magnum Epithelial Cell of Laying Hens

HUANG Xuan-yang，WANG Jian-ping^*，DING Xue-mei，BAI Shi-ping，ZENG Qiu-feng，ZHANG Ke-ying

(Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Key Laboratory for Animal Disease-Resistance Nutrition and Feed of China Ministry of Agriculture, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu 611130, China)

Received:2016-07-29 Online:2017-02-23 Published:2017-02-23

摘要/Abstract

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、1 000 μmol•L^-1）处理12 h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24 h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100 μmol•L^-1的钒处理OMECs 12 h后，细胞活力分别下降到（69.90±2.78）%和（63.60±1.57）%，显著低于25 μmol•L^-1钒处理组（P＜0.05），但是显著高于250和1 000 μmol•L^-1钒处理组（P＜0.05）。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（P＜0.05）；1 000 μmol•L^-1的钒细胞凋亡率为最高（P＜0.05）。4）ROS的测定结果表明，100和250 μmol•L^-1钒处理组的FITC相对平均值显著高于0 μmol•L^-1钒处理组（P＜0.05），且250 μmol•L^-1钒处理组的FITC相对平均值显著高于100 μmol•L^-1钒处理组（P＜0.05）。 5）50 μmol•L^-1钒处理组SOD活性显著低于0 μmol•L^-1钒处理组（P＜0.05），但MDA含量差异不显著。而100 μmol•L^-1钒处理组的MDA含量显著升高（P＜0.05）；50和100 μmol•L^-1钒处理组细胞CAT活性显著低于0 μmol•L^-1钒处理组（P＜0.05）；LDH释放量随着钒的添加剂量的增加而升高，1 000 μmol•L^-1钒处理组LDH的释放量最高，且100 μmol•L^-1钒处理组LDH的释放量显著高于0 μmol•L^-1钒处理组（P＜0.05），达到141.61±2.81 U•gprot^-1。综上表明，OMECs原代细胞培养成功，在OMEC原代细胞里添加100 μmol•L^-1的V⁵⁺处理12 h，可建立OMECs氧化应激模型。

Abstract:

This study was conducted to establish a V⁵⁺-induced oxidative stress model by using primary oviduct magnum epithelial cells (OMECs) of laying hens. OMECs were isolated from Lohman layers (2-3 weeks before onset of laying), cultured and characterized subsequently. Cultured OMECs were then treated with 0, 25, 50, 100, 250, and 1 000 μmol•L^-1 vanadium for 12 h to measure cell viability, apoptosis ratio as well as cellular ROS, SOD, MDA, CAT and LDH released. The results showed that: 1) cell island formed after 24 h and showed classical flagstone morphology. Cultured cells were positive to anti-CK18, anti-OVA, anti-ESR1 and anti-PGR antibody, while were negative to anti-vimentin antibody. 2) Cell viability rate were （69.90±2.78）% and （63.60±1.57）% after 50 and 100 μmol•L^-1 vanadium treated for 12 h, respectively, which were lower (P<0.05) than that of 25 μmol•L^-1 and higher (P<0.05) than that of 250 and 1 000 μmol•L^-1 vanadium. 3) The apoptosis rate increased(P<0.05) as vanadium levels increased, with the 1 000 μmol•L^-1 vanadium treatment had the highest apoptosis rate. 4) The cellular ROS, FITC mean values in 100 and 250 μmol•L^-1 vanadium groups were significantly higher than in 0 μmol•L^-1 vanadium treatment (P<0.05), meanwhile FITC mean value in 250 μmol•L^-1 was significantly higher than in 100 μmol•L^-1 vanadium treatment (P<0.05). 5) Compared with 0 μmol•L^-1 vanadium, cells treated with 50 μmol•L^-1 vanadium had lower (P<0.05) SOD activity, while 100, 250 and 1 000 μmol•L^-1 vanadium groups significantly increased MDA contents(P<0.05). The activity of CAT in 50, 100, 250 and 1 000 μmol•L^-1 vanadium treatments were significantly lower than in 0 μmol•L^-1 vanadium treatment (P<0.05). The amount of released LDH increased as vanadium levels increased, with the 1 000 μmol•L^-1 vanadium treatment had the highest value (P<0.05), and 100 μmol•L^-1 vanadium group had higher value than in 0 μmol•L^-1 (P<0.05), up to 141.61±2.81 U•gprot^-1. These results indicated that the primary OMECs were cultured successfully and supplementation of 100 μmol•L^-1 vanadium in primary OMECs can be used to establish an oxidative stress model in OMECs.

中图分类号:

S831
S85

黄选洋,王建萍,丁雪梅,白世平,曾秋凤,张克英. 钒诱导蛋鸡输卵管膨大部上皮细胞氧化应激模型的建立[J]. 畜牧兽医学报, 2017, 48(2): 340-350.

HUANG Xuan-yang，WANG Jian-ping，DING Xue-mei，BAI Shi-ping，ZENG Qiu-feng，ZHANG Ke-ying. Establishment of an Oxidative Stress Model Induced by Vanadium in Oviduct Magnum Epithelial Cell of Laying Hens[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 340-350.

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钒诱导蛋鸡输卵管膨大部上皮细胞氧化应激模型的建立

Establishment of an Oxidative Stress Model Induced by Vanadium in Oviduct Magnum Epithelial Cell of Laying Hens

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