L-茶氨酸对过氧化氢诱导山羊瘤胃上皮传代细胞凋亡的影响

揭红东, 韩奇鹏, 谭支良, 周传社, 孔志伟, 陈亮, 任傲

doi:10.3969/j.issn.1006-267x.2017.05.017

动物营养学报 >

2017 , Vol. 29 >Issue 5: 1582 - 1589

DOI: https://doi.org/10.3969/j.issn.1006-267x.2017.05.017

分子营养 Molecular Nutrition

L-茶氨酸对过氧化氢诱导山羊瘤胃上皮传代细胞凋亡的影响

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1. 湖南农业大学园艺园林学院, 长沙 410128;
2. 中国科学院亚热带农业生态研究所亚热带农业生态过程重点实验室, 畜禽养殖污染控制与资源化技术国家工程实验室, 湖南省畜禽健康养殖工程技术中心, 农业部中南动物营养与饲料科学观测实验站, 长沙 410125;
3. 湖南畜禽安全生产协同创新中心, 长沙 410128

揭红东(1991-),男,湖南长沙人,硕士研究生,研究方向为药物植物资源工程。E-mail:593432635@qq.com

收稿日期: 2017-01-27

网络出版日期: 2017-05-13

基金资助

国家自然科学基金面上项目（31372342）

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Effects of L-Theanine on Apoptosis of Subculture Ruminal Epithelial Cells Induced by Hydrogen Peroxide of Goats

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1. College of Horticulture & Landscape, Hunan Agricultural University, Changsha 410128, China;
2. Key Laboratory for Agro-Ecological Processes in Subtropical Region, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Hunan Research Center of Livestock & Poultry Sciences, South-Central Experimental Station of Animal Nutrition and Feed Science of Ministry of Agriculture, Institute of Subtropical Agriculture, The Chinese Academy of Science, Changsha 410125, China;
3. Hunan Co-Innovation Center of Animal Production Safety, Changsha 410128, China

Received date: 2017-01-27

Online published: 2017-05-13

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摘要

本试验通过建立过氧化氢（H₂O₂）诱导山羊瘤胃上皮传代细胞凋亡模型，研究L-茶氨酸对H₂O₂诱导山羊瘤胃上皮传代细胞凋亡比率及其凋亡通路关键基因表达量的影响。选取42日龄湘东黑山羊的瘤胃上皮传代细胞，培养于含5%胎牛血清（FBS）的DMEM/F12培养基中，待细胞密度达到60%~70%时，随机分为5组，分别为培养基中无额外添加物的对照组、添加800 μmol/L H₂O₂的Ⅰ组、添加800 μmol/L H₂O₂+4 mmol/L L-茶氨酸的Ⅱ组、添加800 μmol/L H₂O₂+8 mmol/L L-茶氨酸的Ⅲ组和添加800 μmol/L H₂O₂+16 mmol/L L-茶氨酸的Ⅳ组，每组3个重复。作用12 h后，应用流式细胞术（FCM）检测山羊瘤胃上皮传代细胞凋亡比率，并采用实时荧光定量PCR（RT-qPCR）对山羊瘤胃上皮传代细胞凋亡通路关键基因半胱氨酸天冬氨酸-3（Caspase-3）、半胱氨酸天冬氨酸-8（Caspase-8）、半胱氨酸天冬氨酸-9（Caspase-9）、Fas相关死亡域蛋白（FADD）和凋亡酶激活因子（Apaf-1）的表达量进行检测。结果显示：1）通过膜联蛋白-V（annexin V）/碘化丙啶（PI）联合染色结果可知，与Ⅰ组相比，各L-茶氨酸添加组（Ⅱ、Ⅲ和Ⅳ组）细胞晚期凋亡比率显著降低（P<0.05），且细胞晚期凋亡比率随L-茶氨酸添加浓度的增加逐渐降低。2）通过RT-qPCR检测结果可知，与Ⅰ组相比，各L-茶氨酸添加组Caspase-3、Caspase-9、Apaf-1基因的表达量皆显著降低（P<0.05）。由此得出，L-茶氨酸对H₂O₂引起的山羊瘤胃上皮传代细胞凋亡具有一定的保护作用，该结果可为今后研究反刍家畜瘤胃氧化应激损伤机制提供技术支持和理论参考。

关键词： L-茶氨酸; H₂O₂; 山羊; 瘤胃上皮传代细胞; 凋亡

本文引用格式

揭红东, 韩奇鹏, 谭支良, 周传社, 孔志伟, 陈亮, 任傲 . L-茶氨酸对过氧化氢诱导山羊瘤胃上皮传代细胞凋亡的影响[J]. 动物营养学报, 2017 , 29(5) : 1582 -1589 . DOI: 10.3969/j.issn.1006-267x.2017.05.017

Abstract

The aim of this study was to explore the effects of L-theanine on the apoptosis ratio and expression quantities of key genes in apoptosis pathway of subculture ruminal epithelial cells induced by hydrogen peroxide (H₂O₂) of goats according to establish the apoptosis model for subculture ruminal epithelium cells induced by H₂O₂. Subculture ruminal epithelium cells of 42-day-old Xiangdong black goats were selected, and were cultured in DMEM/F12 medium with 5% FBS, when the intensity reached 60% to 70%, the cells were randomly divided into 5 groups: control group, with no supplementation in medium; group Ⅰ, supplemented with 800 μmol/L H₂O₂ in medium; group Ⅱ, supplemented with 800 μmol/L+4 mmol/L L-theanine in medium, group Ⅲ, supplemented with 800 μmol/L+8 mmol/L L-theanine in medium, group Ⅳ, supplemented with 800 μmol/L+16 mmol/L L-theanine in medium. There were three repeats in each group. After 12 h treatment, flow cytometry (FCM) was used to detect the apoptosis ratio of the subculture ruminal epithelium cells, and real-time fluorescent quantitative PCR (RT-qPCR) was selected to detect the expression quantities of apoptosis pathway key genes of subculture ruminal epithelial cells, such as cysteinyl aspartate-specific proteinase-3 (Caspase-3), cysteinyl aspartate-specific proteinase-8 (Caspase-8), cysteinyl aspartate-specific proteinase-9 (Caspase-9), apoptotic protease activating facter-1 (Apaf-1) and Fas-associate with death domain protein (FADD). The results showed as follows: 1) compared with the group Ⅰ, the late apoptosis ratio of cells for L-theanine supplementation groups (groups Ⅱ, Ⅲ and Ⅳ) decreased significantly (P<0.05), and the late apoptosis ratio decreased with the increase of L-theanine supplemental concentration by using annexin V/propidium iodide (PI) unite staining. 2) The expression quantities of Caspase-3, Caspase-9 and Apaf-1 for L-theanine supplementation groups decreased significantly compared with the group Ⅰ using RT-qPCR test (P<0.05). It is concluded that L-theanine plays a protection role in apoptosis subculture ruminal epithelium cells induced by H₂O₂ of goats, which may provide technical support and theoretical reference for the future research on oxidative stress damage mechanism of the rumen in ruminants.

Key words： L-theanine; H₂O₂; goats; subculture ruminal epithelium cell; apoptosis

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