斑马鱼营养性脂肪肝模型构建
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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(q RT-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。
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-monthold 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 q RT-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.
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-monthold 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 q RT-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.
引文
[1]牛化欣,祁飞,贾磊.集约化养殖鱼类脂肪肝疾病及其防治[J].河北渔业,2007(7):47-48.Niu H X,Qi F,Jia L.Intensive aquaculture fish fatty liver disease and its prevention and control[J].Hebei Fisheries,2007(7):47-48(in Chinese).
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[4]Asaoka Y,Terai S,Sakaida I,et al.The expanding role of fish models in understanding non-alcoholic fatty liver disease[J].Disease Models and Mechanisms,2013,6(4):905-914.
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[6]Meguro S,Hasumura T,Hase T.Body fat accumulation in zebrafish is induced by a diet rich in fat and reduced by supplementation with green tea extract[J].PLo S One,2015,10(3):e0120142.
[7]Dai W,Wang K,Zheng X,et al.High fat plus high cholesterol diet lead to hepatic steatosis in zebrafish larvae:a novel model for screening anti-hepatic steatosis drugs[J].Nutrition&Metabolism,2015,12(1):1.
[8]Rio D C,Ares M,Hannon G J,et al.Purification of RNA using TRIzol(TRI reagent)[J].Cold Spring Harbor Protocols,2010(6):5439.
[9]Wang D,Wei Y,Pagliassotti M J.Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis[J].Endocrinology,2006,147(2):943-951.
[10]Li A,Yuan X,Liang X F,et al.Adaptations of lipid metabolism and food intake in response to low and high fat diets in juvenile grass carp(Ctenopharyngodon idellus)[J].Aquaculture,2016,457:43-49.
[11]Lu K L,Xu W N,Li X F,et al.Hepatic triacylglycerol secretion,lipid transport and tissue lipid uptake in blunt snout bream(Megalobrama amblycephala)fed high-fat diet[J].Aquaculture,2013,408:160-168.
[12]Amirkolaie A K,Verreth J A J,Schrama J W.Effect of gelatinization degree and inclusion level of dietary starch on the characteristics of digesta and faeces in Nile tilapia(Oreochromis niloticus L.)[J].Aquaculture,2006,260(1):194-205.
[13]Cani P D,Bibiloni R,Knauf C,et al.Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice[J].Diabetes,2008,57(6):1470-1481.
[14]Ratziu V,Giral P,Charlotte F,et al.Liver fibrosis in overweight patients[J].Gastroenterology,2000,118(6):1117-1123.
[15]张介眉,郝建军,时昭红,等.华夏小葱制剂对脂肪肝大鼠脂质代谢和组织形态学的影响[J].世界华人消化杂志,2007,15(22):2447-2452.Zhang J M,Hao J J,Shi S H,et al.effect of a preparation of chinese Allium fistulosum L on the morphology and metabolism of lipids in fatty liver rats[J].World Chinese Journal of Digestology,2007,15(22):2447-2452(in Chinese).
[16]Smith S J,Cases S,Jensen D R,et al.Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat[J].Nature Genetics,2000,25(1):87-90.
[17]Monetti M,Levin M C,Watt M J,et al.Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver[J].Cell Metabolism,2007,6(1):69-78.
[18]Ferre P,Foufelle F,et al.Hepatic steatosis:a role for de novo lipogenesis and the transcription factor SREBP-1c[J].Diabetes,Obesity and Metabolism,2010,12(s2):83-92.
[19]Rosen E D,Sarraf P,Troy A E,et al.PPARγis required for the differentiation of adipose tissue in vivo and in vitro[J].Molecular Cell,1999,4(4):611-617.
[20]Tontonoz P,Nagy L,Alvarez J G A,et al.PPARγpromotes monocyte/macrophage differentiation and uptake of oxidized LDL[J].Cell,1998,93(2):241-252.
[21]Ito M,Nagasawa M,Hara T,et al.Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes[J].Journal of Lipid Research,2010,51(7):1676-1684.
[22]Ye J,Li J Z,Liu Y,et al.Cideb,an ER-and lipid dropletassociated protein,mediates VLDL lipidation and maturation by interacting with apolipoprotein B[J].Cell Metabolism,2009,9(2):177-190.
[23]Bézaire V,Mairal A,Anesia R,et al.Chronic TNFαand c AMP pre-treatment of human adipocytes alter HSL,ATGL and perilipin to regulate basal and stimulated lipolysis[J].FEBS Letters,2009,583(18):3045-3049.
[24]Iseri O A,Lieber C S,Gottlieb L S.The ultrastructure of fatty liver induced by prolonged ethanol ingestion[J].The American Journal of Pathology,1966,48(4):535.
[25]郭晓霞,李良学,武玉鹏,等.调肝理脾方干预原发性胆汁性肝硬化小鼠肝损伤的组织学观察[J].中西医结合肝病杂志,2015,25(1):36-38,67.Guo X X,Li L X,Wu Y P,et al.Intervention action of Tiaogan Lipi prescription on hepatic tissue of mouse with primary biliary cirrhosis[J].Chinese Journal of Integrated Traditonal and Western Medicine on Liver Diseases,2015,25(1):36-38,67(in Chinese).
[26]王静香,李加儿,区又君,等.驼背鲈肝脏结构的光镜和透射电镜观察[J].海洋渔业,2010,32(4):388-394.Wang J X,Li J E,Qu Y J,et al.Observation on light and transmission electron microscopy of liver structure in high finned grouper Cromileptes altivelis[J].Marine Fisheries,2010,32(4):388-394(in Chinese).
[27]Reaven E P,Peterson D T,Reaven G M.The effect of experimental diabetes mellitus and insulin replacement on hepatic ultrastructure and protein synthesis[J].Journal of Clinical Investigation,1973,52(2):248.
[28]PlanagumàA,Clària J,Miquel R,et al.The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPARγactivation[J].The FASEB Journal,2005,19(9):1120-1122.
[29]韩芳.PPARγ基因修饰对非酒精性脂肪性肝纤维化的防治机制[D].石家庄:河北医科大学,2010.Han F.Targeted PPARγactivation prevents non-alcoholic fibrotic steatohepatitis in mice[D].Shijiazhuang:Hebei Medical University,2010(in Chinese).
[30]徐正婕,范建高,王国良,等.高脂饮食致大鼠非酒精性脂肪性肝炎肝纤维化模型[J].世界华人消化杂志,2002(4):392-396.Xu Z J,Fan J G,Wang G L,et al.Rat model of nonalcoholic steatohepatitis with fibrosis by a fat-rich diet[J].World Chinese Journal of Dig,2002(4):392-396(in Chinese).
[2]杨鸿昆,黄凯,阮栋俭.养殖鱼类脂肪肝及防治研究进展[J].水利渔业,2007,27(1):4-6.Yang H K,Huang K,Ruan D J.Farmed fish fatty liver disease and the research progress of prevention and cure[J].Reservoir Fisheries,2007,27(1):4-6(in Chinese).
[3]杜震宇.养殖鱼类脂肪肝成因及相关思考[J].水产学报,2014,38(9):1628-1638.Du Z Y.Causes of fatty liver in farmed fish:a review and new perspectives[J].Journal of Fisheries of China,2014,38(9):1628-1638(in Chinese).
[4]Asaoka Y,Terai S,Sakaida I,et al.The expanding role of fish models in understanding non-alcoholic fatty liver disease[J].Disease Models and Mechanisms,2013,6(4):905-914.
[5]Shimada Y,Kuninaga S,Ariyoshi M,et al.E2F8promotes hepatic steatosis through FABP3 expression in diet-induced obesity in zebrafish[J].Nutrition&Metabolism,2015,12(1):1.
[6]Meguro S,Hasumura T,Hase T.Body fat accumulation in zebrafish is induced by a diet rich in fat and reduced by supplementation with green tea extract[J].PLo S One,2015,10(3):e0120142.
[7]Dai W,Wang K,Zheng X,et al.High fat plus high cholesterol diet lead to hepatic steatosis in zebrafish larvae:a novel model for screening anti-hepatic steatosis drugs[J].Nutrition&Metabolism,2015,12(1):1.
[8]Rio D C,Ares M,Hannon G J,et al.Purification of RNA using TRIzol(TRI reagent)[J].Cold Spring Harbor Protocols,2010(6):5439.
[9]Wang D,Wei Y,Pagliassotti M J.Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis[J].Endocrinology,2006,147(2):943-951.
[10]Li A,Yuan X,Liang X F,et al.Adaptations of lipid metabolism and food intake in response to low and high fat diets in juvenile grass carp(Ctenopharyngodon idellus)[J].Aquaculture,2016,457:43-49.
[11]Lu K L,Xu W N,Li X F,et al.Hepatic triacylglycerol secretion,lipid transport and tissue lipid uptake in blunt snout bream(Megalobrama amblycephala)fed high-fat diet[J].Aquaculture,2013,408:160-168.
[12]Amirkolaie A K,Verreth J A J,Schrama J W.Effect of gelatinization degree and inclusion level of dietary starch on the characteristics of digesta and faeces in Nile tilapia(Oreochromis niloticus L.)[J].Aquaculture,2006,260(1):194-205.
[13]Cani P D,Bibiloni R,Knauf C,et al.Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice[J].Diabetes,2008,57(6):1470-1481.
[14]Ratziu V,Giral P,Charlotte F,et al.Liver fibrosis in overweight patients[J].Gastroenterology,2000,118(6):1117-1123.
[15]张介眉,郝建军,时昭红,等.华夏小葱制剂对脂肪肝大鼠脂质代谢和组织形态学的影响[J].世界华人消化杂志,2007,15(22):2447-2452.Zhang J M,Hao J J,Shi S H,et al.effect of a preparation of chinese Allium fistulosum L on the morphology and metabolism of lipids in fatty liver rats[J].World Chinese Journal of Digestology,2007,15(22):2447-2452(in Chinese).
[16]Smith S J,Cases S,Jensen D R,et al.Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat[J].Nature Genetics,2000,25(1):87-90.
[17]Monetti M,Levin M C,Watt M J,et al.Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver[J].Cell Metabolism,2007,6(1):69-78.
[18]Ferre P,Foufelle F,et al.Hepatic steatosis:a role for de novo lipogenesis and the transcription factor SREBP-1c[J].Diabetes,Obesity and Metabolism,2010,12(s2):83-92.
[19]Rosen E D,Sarraf P,Troy A E,et al.PPARγis required for the differentiation of adipose tissue in vivo and in vitro[J].Molecular Cell,1999,4(4):611-617.
[20]Tontonoz P,Nagy L,Alvarez J G A,et al.PPARγpromotes monocyte/macrophage differentiation and uptake of oxidized LDL[J].Cell,1998,93(2):241-252.
[21]Ito M,Nagasawa M,Hara T,et al.Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes[J].Journal of Lipid Research,2010,51(7):1676-1684.
[22]Ye J,Li J Z,Liu Y,et al.Cideb,an ER-and lipid dropletassociated protein,mediates VLDL lipidation and maturation by interacting with apolipoprotein B[J].Cell Metabolism,2009,9(2):177-190.
[23]Bézaire V,Mairal A,Anesia R,et al.Chronic TNFαand c AMP pre-treatment of human adipocytes alter HSL,ATGL and perilipin to regulate basal and stimulated lipolysis[J].FEBS Letters,2009,583(18):3045-3049.
[24]Iseri O A,Lieber C S,Gottlieb L S.The ultrastructure of fatty liver induced by prolonged ethanol ingestion[J].The American Journal of Pathology,1966,48(4):535.
[25]郭晓霞,李良学,武玉鹏,等.调肝理脾方干预原发性胆汁性肝硬化小鼠肝损伤的组织学观察[J].中西医结合肝病杂志,2015,25(1):36-38,67.Guo X X,Li L X,Wu Y P,et al.Intervention action of Tiaogan Lipi prescription on hepatic tissue of mouse with primary biliary cirrhosis[J].Chinese Journal of Integrated Traditonal and Western Medicine on Liver Diseases,2015,25(1):36-38,67(in Chinese).
[26]王静香,李加儿,区又君,等.驼背鲈肝脏结构的光镜和透射电镜观察[J].海洋渔业,2010,32(4):388-394.Wang J X,Li J E,Qu Y J,et al.Observation on light and transmission electron microscopy of liver structure in high finned grouper Cromileptes altivelis[J].Marine Fisheries,2010,32(4):388-394(in Chinese).
[27]Reaven E P,Peterson D T,Reaven G M.The effect of experimental diabetes mellitus and insulin replacement on hepatic ultrastructure and protein synthesis[J].Journal of Clinical Investigation,1973,52(2):248.
[28]PlanagumàA,Clària J,Miquel R,et al.The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPARγactivation[J].The FASEB Journal,2005,19(9):1120-1122.
[29]韩芳.PPARγ基因修饰对非酒精性脂肪性肝纤维化的防治机制[D].石家庄:河北医科大学,2010.Han F.Targeted PPARγactivation prevents non-alcoholic fibrotic steatohepatitis in mice[D].Shijiazhuang:Hebei Medical University,2010(in Chinese).
[30]徐正婕,范建高,王国良,等.高脂饮食致大鼠非酒精性脂肪性肝炎肝纤维化模型[J].世界华人消化杂志,2002(4):392-396.Xu Z J,Fan J G,Wang G L,et al.Rat model of nonalcoholic steatohepatitis with fibrosis by a fat-rich diet[J].World Chinese Journal of Dig,2002(4):392-396(in Chinese).