动物营养学报 >

2020 , Vol. 32 >Issue 8: 3830 - 3842

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.08.042

分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

铁水平和孵育时间对原代培养肉鸡鸡胚肝细胞中铁含量及含铁酶活性、基因表达和蛋白表达的影响

  • 马雪莲 ,
  • 廖秀冬 ,
  • 张丽阳 ,
  • 吕林 ,
  • 罗绪刚
展开
  • 1. 中国农业科学院北京畜牧兽医研究所, 矿物元素营养研究室, 北京 100193;
    2. 扬州大学动物科技学院, 家禽矿物元素营养研究室, 扬州 225000
马雪莲(1993-),女,甘肃天水人,硕士研究生,从事微量元素营养研究。E-mail:963243900@qq.com

收稿日期: 2020-03-13

  网络出版日期: 2020-08-13

基金资助

国家自然科学基金项目（31672440）；国家现代农业产业技术体系岗位专家专项经费（CARS-41）；中国农业科学院科技创新工程专项经费（ASTIP-IAS08）

收起

Effects of Iron Level and Incubation Time on Iron Content and Activity, Gene Expression and Protein Expression of Iron-Containing Enzyme in Primarily Cultured Hepatocytes of Broiler Embryos

  • MA Xuelian ,
  • LIAO Xiudong ,
  • ZHANG Liyang ,
  • LYU Lin ,
  • LUO Xugang
Expand
  • 1. Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. Poultry Mineral Nutrition Laboratory, College of Animal Science and Technology, Yangzhou University, Yangzhou 225000, China

Received date: 2020-03-13

  Online published: 2020-08-13

Fold

摘要

本试验旨在研究铁水平和孵育时间对原代培养肉鸡鸡胚肝细胞中铁含量及含铁酶活性、基因表达和蛋白表达的影响。采用5×3两因子完全随机设计，铁水平分别为0、0.25、0.50、0.75、1.00 mmol/L，孵育时间分别为24、48和72 h，共形成15个组，每组6个重复。结果表明：1）铁水平、孵育时间及两者的互作对肝细胞铁含量有显著影响（P<0.05）。24、48和72 h时，0.25、0.50、0.75、1.00 mmol/L铁水平组肝细胞中铁含量均显著高于0 mmol/L铁水平组（P<0.05）。2）铁水平和孵育时间对肝细胞中胞琥珀酸脱氢酶（SDH）和细胞色素c氧化酶（COX）活性有显著影响（P<0.05）。与0 mmol/L铁水平组相比，0.25、0.50、0.75和1.00 mmol/L铁水平组肝细胞中SDH活性显著降低（P<0.05）；24 h肝细胞中SDH活性显著高于48 h（P<0.05），而72 h又显著高于24和48 h（P<0.05）。与0 mmol/L铁水平组相比，0.75和1.00 mmol/L铁水平组肝细胞中COX活性显著提高（P<0.05）；24和48 h肝细胞中COX活性显著高于72 h（P<0.05）。3）铁水平、孵育时间及两者的互作对肝细胞中SDH、过氧化氢酶（CAT）、COX7A2L和铁蛋白重链1（FTH1）的mRNA表达水平有显著影响（P<0.05），铁水平及铁水平与孵育时间的互作对肝细胞中COX1的mRNA表达水平有显著影响（P<0.05）。4）铁水平和孵育时间对肝细胞中COX1的蛋白表达水平有显著影响（P<0.05），铁水平及铁水平与孵育时间的互作对肝细胞中FTH1的蛋白表达水平表达有显著影响（P<0.05）。24和72 h肝细胞中COX1的蛋白表达水平显著高于48 h（P<0.05），1.00 mmol/L铁水平组肝细胞中COX1的蛋白表达水平显著高于其他铁水平组（P<0.05）。由此可见，铁水平和孵育时间可影响原代培养肉鸡鸡胚肝细胞中铁含量，SDH、CAT和COX的活性、基因表达，以及FTH1的基因表达。综合考虑以上指标，适宜铁的水平应为0.50 mmol/L，孵育时间为24 h。

关键词： 原代培养肝细胞; 肉鸡鸡胚; 铁; 含铁酶; 基因表达

本文引用格式

马雪莲 , 廖秀冬 , 张丽阳 , 吕林 , 罗绪刚 . 铁水平和孵育时间对原代培养肉鸡鸡胚肝细胞中铁含量及含铁酶活性、基因表达和蛋白表达的影响[J]. 动物营养学报, 2020 , 32(8) : 3830 -3842 . DOI: 10.3969/j.issn.1006-267x.2020.08.042

Abstract

This experiment was conducted to investigate the effects of iron (Fe) level and incubation time on Fe concentration, Fe-containing enzyme activity, gene expression and protein expression in primary cultured hepatocytes of broiler embryos. A 5×3 two factor completely randomized design was used in the experiment, the Fe levels were 0, 0.25, 0.50, 0.75 and 1.00 mmol/L, respectively, and the incubation times were 12, 24 and 48 h, respectively. There were assigned to 15 groups with 6 replicates per group. The result showed as follows:1) Fe level, incubation time and there interaction had significant effects on content of Fe in hepatocytes (P<0.05). At 24, 48 and 72 h, the content of Fe in hepatocytes of 0.25, 0.50, 0.75, 1.00 mmol/L Fe level groups was significantly higher than that of 0 mmol/L Fe level group (P<0.05). 2) Fe level and incubation time had significant effects on activities of succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) in hepatocytes (P<0.05). Compared with the 0 mmol/L Fe level group, the activity of SDH in hepatocytes of 0.25, 0.50, 0.75 and 1.00 mmol/L Fe level groups was significantly decreased (P<0.05); the activity of SDH in hepatocytes at 24 h was significantly higher than that at 48 h (P<0.05), and 72 h was significantly higher than 24 and 48 h (P<0.05). Compared with the 0 mmol/L Fe level group, the activity of COX in hepatocytes of 0.75 and 1.00 mmol/L Fe level groups was significantly increased (P<0.05); the activity of COX in hepatocytes at 24 and 48 h was significantly higher than that at 72 h (P<0.05). 3) Fe level, incubation time and there interaction had significant effects on mRNA expression levels of SDH, catalase (CAT), COX7A2L, ferritin heavy chain 1 (FTH1) in hepatocytes (P<0.05), and Fe level and interaction of Fe level and incubation time had significant effects on mRNA expression level of COX1 in hepatocytes (P<0.05). 4) Fe level and incubation time had significant effects on protein expression level of COX1 in hepatocytes (P<0.05), and Fe level and interaction of Fe level and incubation time had significant effects on protein expression level of FTH1 in hepatocytes (P<0.05). The protein expression level of COX1 in hepatocytes at 24 and 72 h was significantly higher than that at 48 h (P<0.05), and the protein expression level of COX1 in hepatocytes of 1.00 mmol/L Fe level group was significantly higher than that of other Fe level groups (P<0.05). In conclusion, Fe level and incubation time can affect the Fe content, activities and gene expression of SDH, CAT and COX, and the gene expression of FTH1 in primary cultured hepatocytes of broiler embryos. Consider the above indicators comprehensively, the optimal Fe level is 0.50 mmol/L, and the optimal incubation time is 24 h.

Key words： primary cultured hepatocytes; broiler embryos; iron; iron-containing enzyme; gene expression

参考文献

[1] CRICHTON R R,CHARLOTEAUX-WAUTERS M.Iron storage and transport[J].European Journal of Biochemitry,1987,164(3):485-506.  
[2] THEIL E C.Ferritin:structure,gene regulation,and cellular function in animals,plants,and microorganisms[J].Annual Review of Biochemistry,1987,56(1):289-315.  
[3] QUINTANA C,BELLEFQIH S,LAVAL J Y,et al.Study of the localization of iron,ferritin,and hemosiderin in Alzheimer's disease hippocampus by analytical microscopy at the subcellular level[J].Journal of Structural Biology,2006,153(1):42-54.  
[4] GLAHN R P,CHENG Z Q,WELCH R M,et al.Comparison of iron bioavailability from 15 rice genotypes:studies using an in vitro digestion/caco-2 cell culture model[J].Journal of Agricultural and Food Chemistry,2002,50(12):3586-3591.  
[5] KLOOTS W,KAMP D O D,ABRAHAMSE L.In vitro iron availability from iron-fortified whole-grain wheat flour[J].Journal of Agricultural and Food Chemistry,2004,52(26):8132-8136.  
[6] BROCK J H.Iron in infection,immunity,inflammation and neoplasia[Z]//BROCK J H,HALLIDAY J W,PIPPARD M J,et al.Iron metabolism in health and disease.London:Saunders,1994:354-389.
[7] ALIBEGOVIC F,KAVAZOVIC A,GAGIC A,et al.The effect of dietary iron on its content in the liver in broilers[J].Veterinaria,2011:113-120.
[8] 张银银,王冬尧,沈慧,等.铁代谢紊乱与肝癌的研究进展[J].生命科学,2019,31(11):1148-1157.
[9] SHEFTEL A,STEHLING O,LILL R.Iron-sulfur proteins in health and disease[J].Trends in Endocrinology & Metabolism,2010,21(5):302-314.  
[10] GOZZELINO R,JENEY V,SOARES M P.Mechanisms of cell protection by heme oxygenase-1[J].Annual Review of Pharmacology and Toxicology,2010,50(1):323-354.  
[11] TAKO E,RUTZKE M A,GLAHN R P.Using the domestic chicken (Gallus gallus) as an in vivo model for iron bioavailability[J].Poultry Science,2010,89(3):514-521.  
[12] EVSTATIEV R,GASCHE C.Iron sensing and signaling[J].Gut,2012,61(6):933-952.  
[13] DE DEUNGRIA M,RAO R,WOBKEN J D,et al.Perinatal iron deficiency decreases cytochrome c oxidase (CytOx) activity in selected regions of neonatal rat brain[J].Pediatric Research,2000,48(2):169-176.  
[14] MCKIE A T,BARROW D,LATUNDE-DADA G O.An iron-regulated ferric reductase associated with the absorption of dietary iron[J].Science,2001,291(5509):1755-1759.  
[15] FENG J,MA W Q,XU Z R,et al.The effect of iron glycine chelate on tissue mineral levels,fecal mineral concentration,and liver antioxidant enzyme activity in weanling pigs[J].Animal Feed Science and Technology,2009,150(1/2):106-113.
[16] NAVARRO A,BOVERIS A.The mitochondrial energy transduction system and the aging process[J].American Journal of Physiology:Cell Physiology,2007,292(2):C670-C686.
[17] ZHANG L Y,LU L,ZHANG L Y,et al.The chemical characteristics of organic iron sources and their relative bioavailabilities for broilers fed a conventional corn-soybean meal diet[J].Journal of Animal Science,2016,94(6):2378-2396.  
[18] MA X Y,LIAO X D,LU L,et al.Determination of dietary iron requirements by full expression of iron-containing enzymes in various tissues of broilers[J].The Journal of Nutrition,2016,146(11):2267-2273.  
[19] BALLA G,JACOB H S,BALLA J,et al.Ferritin:a cytoprotective antioxidant strategem of endothelium[J].Journal of Biological Chemistry,1992,267(25):18148-18153.
[20] ROGERS J,MUNRO H.Translation of ferritin light and heavy subunit mRNAs is regulated by intracellular chelatable iron levels in rat hepatoma cells[J].Proceedings of the National Academy Sciences of the United States of America,1987,84(8):2277-2281.  
[21] AHMAD S,SULTAN S,NAZ N,et al.Regulation of iron uptake in primary culture rat hepatocytes;the role of acute-phase cytokines[J].Shock,2014,41(4):337-345.  
[22] 王美玲.锰对原代培养肉鸡肝细胞脂肪合成关键酶活性及其基因表达的影响[D].硕士学位论文.北京:中国农业科学院,2011.
[23] 何文刚.锌对原代培养肉鸡鸡胚热敏肝细胞的抗热应激效应及其分子机制研究[D].硕士学位论文.兰州:甘肃农业大学,2017.
[24] GOKDUMAN K,BESTEPE F,LI L,et al.Dose-,treatment-and time-dependent toxicity of superparamagnetic iron oxide nanoparticles on primary rat hepatocytes[J].Nanomedicine,2018,13(11):1267-1284.  
[25] ZHANG L Y,LI X F,LIAO X D,et al.Effect of iron source on iron absorption and gene expression of iron transporters in the ligated duodenal loops of broilers[J].Journal of Animal Science,2017,95(4):1587-1597.
[26] MA X Y,LIU S B,LU L,et al.Relative bioavailability of iron proteinate for broilers fed a casein-dextrose diet[J].Poultry Science,2014,93(3):556-563.  
[27] SEOL S H,LEE H K,LEE W S,et al.The effect of level and period of Fe-methionine chelate supplementation on the iron content of boiler meat[J].Asian-Australian Journal of Animal Science,2008,21(10):1501-1505.  
[28] SHI Z H,NIE G J,DUAN X L,et al.Neuroprotective mechanism of mitochondrial ferritin on 6-hydroxydopamine-induced dopaminergic cell damage:implication for neuroprotection in Parkinson's disease[J].Antioxidants & Redox Signaling,2010,13(6):783-796.  
[29] HINTZE K J,THEIL E C.Cellular regulation and molecular interactions of the ferritins[J].Cellular and Molecular Life Sciences,2006,63(5):591-600.  
[30] GHOSH K.Non haematological effects of iron deficiency-a perspective[J].Indian Journal of Medical Sciences,2006,60(1):30-37.  
[31] SALETTA F,RAHMANTO Y S,NOULSRI E,et al.Iron chelator-mediated alterations in gene expression:identification of novel iron-regulated molecules that are molecular targets of hypoxia-inducible factor-1α and p53[J].Molecular Pharmacology,2010,77(3):443-458.  
[32] 张兆琴,任文陟,张嘉保,等.添加不同水平铁对吉戎Ⅱ系獭兔血液酶活力影响的研究[J].吉林农业大学学报,2006,28(4):433-435,439.
[33] LIAO X D,MA C Y,LU L,et al.Determination of dietary iron requirements by full expression of iron-containing cytochrome c oxidase in the heart of broilers from 22 to 42 d of age[J].British Journal of Nutrition,2017,118(7):493-499.  
[34] 陈静,赵永亮,王丹.铁蛋白研究现状[J].河南师范大学学报(自然科学版),2010,38(1):152-155.
[35] HENTZE M W,MUCKENTHALER M U,GALY B,et al.Two to tango:regulation of mammalian iron metabolism[J].Cell,2010,142(1):24-38.  
[36] THOMSON A M,ROGERS J T,LEEDMAN P J.Iron-regulatory proteins,iron-responsive elements and ferritin mRNA translation[J].The International Journal of Biochemistry & Cell Biology,1999,31(10):1139-1152.  
[37] DRAKESMITH H,NEMETH E,GANZ T.Ironing out ferroportin[J].Cell Metabolism,2015,22(5):777-787.  
[38] GANZ T.Cellular iron:ferroportin is the only way out[J].Cell Metabolism,2005,1(3):155-157.  
Options
文章导航

/

〈 〉

    联系我们

  • 地址：北京市海淀区圆明园西路2号
    　　　中国农业大学动科动医大楼153室
  • 邮编：100193
  • 电话：010－62817823
  • 网址：http://www.chinajan.com
  • E-mail：cjan@chinajan.com
京公网安备 11010802041926号    京ICP备17050989号-2