文章摘要
李钰,何夙旭,张震,徐俐,丁倩雯,郭小泽,易敢峰,徐树德,马徐发,周志刚.斑马鱼营养性脂肪肝模型构建[J].水产学报,2017,41(5):775~784
斑马鱼营养性脂肪肝模型构建
Establishment of Danio rerio nutritional induced fatty liver model
投稿时间:2016-08-24  修订日期:2014-10-16
DOI:10.11964/jfc.20160810513
中文关键词: 斑马鱼  高脂饲料  鱼类脂肪肝  肝纤维化
英文关键词: Danio rerio  high fat diet  fatty liver of fish  liver fibrosis
基金项目:国家自然科学基金(31272672,31572633)
作者单位E-mail
李钰 华中农业大学水产学院, 湖北 武汉 430072
中国农业科学院饲料研究所, 北京 100081 
 
何夙旭 中国农业科学院饲料研究所, 北京 100081  
张震 中国农业科学院饲料研究所, 北京 100081  
徐俐 中国农业科学院饲料研究所, 北京 100081  
丁倩雯 中国农业科学院饲料研究所, 北京 100081  
郭小泽 中国农业科学院饲料研究所, 北京 100081  
易敢峰 福建大北农水产科技有限公司, 福建 漳州 363500  
徐树德 广东溢多利生物科技股份有限公司, 广东 珠海 519060  
马徐发 华中农业大学水产学院, 湖北 武汉 430072 xufama@mail.hzau.edu.cn 
周志刚 中国农业科学院饲料研究所, 北京 100081 zhou_zg@msn.com 
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中文摘要:
      近年来我国集约化水产养殖业发展势头强劲,而水产动物营养性疾病之鱼类脂肪肝的发病也逐渐迅猛起来,逐渐成为制约水产养殖业发展的因素之一,实验拟通过高脂饲料诱导建立斑马鱼营养性脂肪肝模型,为鱼类营养性脂肪肝相关研究工作的开展奠定基础。该实验分别以基础饲料和16%高脂饲料投喂1月龄斑马鱼,在投喂后第14、30、90天3个时间点取样,采用肝脏组织进行H.E染色、透射电镜、masson染色组织切片观察斑马鱼肝脏脂肪变性及纤维化程度;通过荧光定量PCR技术(qRT-PCR)检测斑马鱼肝脏脂合成及脂解相关基因:二酰甘油酰基转移酶基因(DGAT)、过氧化物酶体增殖物激活受体基因(PPAR)、胆固醇调节元件1基因(SREBP1)、脂肪酸合成酶基因(FAS)、诱导细胞死亡脱氧核糖核酸裂解因子样效应因子基因(CIDE)、激素敏感脂肪酶基因(HSL)、甘油三酯水解酶基因(ATGL)的表达水平;采用甲醇–氯仿法提取斑马鱼肝脏脂肪并检测肝脏中甘油三酯的含量。结果显示,在高脂饲料诱导第30天,光镜下可见发生弥漫性肝细胞脂肪变性,脂合成基因显著上调,甘油三脂的含量显著增加;诱导第90天,经透射电镜及masson染色组织切片观察结果证实,斑马鱼肝脏组织由于纤维化病变而使得肝组织功能受损,肝脏的脂合成基因及甘油三脂含量显著降低。研究表明,实验设计的高脂饲料对肝脏的损伤作用显著,30 d的投喂即可诱导发生脂肪肝,90 d投喂斑马鱼肝脏出现肝纤维化,此过程中肝脏脂合成基因表达量及甘油三酯含量先升高后降低,可知斑马鱼肝脏的损伤是由高脂饲料诱导的脂肪大量堆积而引起的,该营养性模型的成功构建在一定程度上弥补了以往构建斑马鱼高脂模型的缺陷,为研究鱼类营养性脂肪肝的发生机制提供了支撑。
英文摘要:
      With the rapid development of aquaculture in recent years, the incidence of aquatic animal diseases, especially fatty liver disease, and its prevalence tended to increase and gradually became a stumbling block of aquaculture industry. Due to lack of effective model to study the pathogenesis, we intend to establish a fatty liver model of zebrafish induced by high-fat diet. In this study, normal diet and 16% high fat diet were fed to 1-month-old zebrafish, respectively. After 14, 30 and 90 days feeding, liver was taken as sample to conduct HE staining, transmission electron microscopy (SEM) and masson staining, and qRT-PCR was used to detect liver tissue genes expression level related to lipogenesis and lipolysis such as DGAT, PPARγ, FAS, SREBP1, CIDE, HSL, ATGL, and through Methanol-chloroform extraction we also detected the liver tissue contents of triglyceride. The result has shown, on the 30 th day, hepatic steatosis has occurred, the lipogenesis genes expression and the contents of triglyceride were significantly increased in high fat diet group. On the 90 th day, high fat diet group of zebrafish liver transmission electron microscopy (sem) and masson staining tissue biopsy showed zebrafish liver fibrosis and liver damage, and liver lipid synthesis genes and triglycerides contents decreased significantly. In conclusion, our high fat diet is able to induce hepatic injury, after 30 d feeding, zebrafish got hepatic steatosis and after 90 d feeding, liver fibrosis happened. Its fat synthetic gene expression level and contents of triglyceride in liver rose first and then fell, which has proven that the fat accumulation induced hepatic injury. The construction of the model remedied the defects of previously-built zebrafish high-fat model and this model will provide a support to study the mechanism of fish nutritional fatty liver disease.
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