L-茶氨酸对过氧化氢诱导山羊瘤胃上皮细胞凋亡的保护作用  

Protection of L-Theanine on Apoptosis of Rumen Epithelial Cells Induced by Hydrogen Peroxide H_2O_2 in Goats

作　　者：孔志伟 揭红东[3] 陈亮 任傲[3] 周传社 谭支良[2,4] KONG Zhiwei 1,2 , JIE Hongdong 3 ,CHEN Liang 3 ,REN Ao 3 ,ZHOU Chuanshe 2,4, TAN Zhiliang 2,4(1. University of the Chinese Academy of Sciences, Beijing 100049, 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, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Hunan Research Center of Livestock ＆ Poultry Sciences, Institute of Subtropical Agriculture, The Chinese Academy of Sciences,Changsha 410125, China; 3. College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China; 4. Hunan Co-Innovation Center of Safety Animal Production, Changsha, Hunan 410128, Chin)

机构地区：[1]中国科学院大学,北京100049 [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]湖南农业大学动物科技学院,长沙410128 [4]湖南畜禽安全生产协同创新中心,长沙410128

出　　处：《动物营养学报》2018年第8期3125-3133,共9页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金面上项目(31772632;31372342);湖南省杰青(2017JJ028);中国科学院青年创新促进会课题(2015302);中国科学院亚热带农业生态研究所青年创新团队项目(2017QNCXTD_ZCS)

摘　　要：本试验旨在研究L-茶氨酸对过氧化氢(H_2O_2)诱导山羊瘤胃上皮细胞凋亡的保护作用。采集42日龄的湘东黑山羊的瘤胃组织进行原代细胞分离、培养、纯化。对照组采用完全培养基,Ⅰ、Ⅱ、Ⅲ、Ⅳ组分别在完全培养基中添加800μmol/L H_2O_2、4 mmol/L L-茶氨酸+800μmol/L H_2O_2、8 mmol/L L-茶氨酸+800μmol/L H_2O_2和16 mmol/L L-茶氨酸+800μmol/L H_2O_2,每组3个重复。培养12 h后,采用噻唑蓝(MTT)法检测细胞存活率,采用流式细胞术检测细胞周期,采用实时定量PCR法和蛋白质印迹(Western blot)法检测Bax和Bcl-2基因及其蛋白的表达。结果表明:L-茶氨酸能显著提高H_2O_2损伤的瘤胃上皮细胞存活率(P<0.05),显著增加S期细胞比例(P<0.05),显著降低Bax基因和蛋白表达量(P<0.05),显著提高Bcl-2基因和蛋白表达量(P<0.05),显著提高Bcl-2和Bax基因和蛋白表达量的比值(P<0.05)。结果提示,L-茶氨酸能通过抑制细胞凋亡来有效地保护由H_2O_2导致的瘤胃上皮细胞损伤;体外条件下,培养液中添加的L-茶氨酸浓度高于8 mm/L时能有效发挥抗凋亡作用。对瘤胃上皮细胞损伤有一定的治疗和保护作用。The object of this experiment was to explore the protection of L -theanine on apoptosis of rumen epithelial cells induced by hydrogen peroxide （H 2O 2） in goats. Ruminal epithelial tissue from Xiangdong black goats aged 42 days were selected for isolation, cultivation and purification of primary cells. The cells were cultured in a complete culture medium in control group, and those in groups Ⅰ, Ⅱ, Ⅲ and Ⅳ were cultured in the complete culture medium supplemented with 800 μmol/L H 2O 2, 4 mmol/L L -theanine＋800 μmol/L H 2O 2, 8 mmol/L L -theanine＋800 μmol/L H 2O 2, and 16 mmol/L L -theanine ＋ 800 μmol/L H 2O 2, respectively. Each group had 3 replicates. After 12 h culture, thiazolyl blue （MTT） method was used to detect the cells viability, flow cytometry （FCM） was used to detect the cell cycle, real-time quantitative PCR and Western blot method were used to detect expressions of Bcl -2 and Bax genes and their proteins. The results showed that L -theanine significantly attenuated the cell viability loss （ P 〈0.05）, significantly increased the cell ratio in S phrase （ P 〈0.05）, significantly decreased expression levels of Bax gene and protein （ P 〈0.05）, significantly increased expression levels of Bcl-2 gene and protein （ P 〈0.05）, and significantly increased the ratios of Bax gene to Bcl-2 gene and Bax protein to Bcl -2 protein in rumen epithelial cells injured by H 2O 2 （ P 〈0.05）. The results indicate that L -theanine can prevent rumen epithelial cells from H 2O 2-induced cell apoptosis. Under in vitro conditions, the supplementation of L -theanine more than 8 mm/L can effectively help to protect the rumen epithelial cells from apoptosis.

关 键 词：L-茶氨酸 过氧化氢 瘤胃上皮细胞 凋亡 

分 类 号：S816.7[农业科学—饲料科学]