动物营养学报 >

2012 , Vol. 24 >Issue 3: 487 - 496

DOI: https://doi.org/10.3969/j.issn.1006-267x.2012.03.015

分子营养

苏氨酸对体外培养感染伪狂犬病毒猪空肠上皮细胞免疫相关基因表达的影响

  • 韩国全 ,
  • 余冰 ,
  • 陈代文 ,
  • 相振田 ,
  • 陈渝 ,
  • 陈洪 ,
  • 毛倩
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  • 1. 四川农业大学动物营养研究所,雅安 625014;
    2. 四川农业大学动物抗病营养教育部重点实验室,雅安 625014

收稿日期: 2011-09-27

  网络出版日期: 2012-02-28

基金资助

教育部长江学者和创新团队发展计划项目资助(IRTO555);现代农业产业技术体系建设专项资金资助(CARS-36);博士后资金资助(04310610)

收起

Effects of Threonine on Expression of Immune-Related Genes of Porcine Intestinal Epithelial Cell Infected with Pseudorabies Virus in Vitro

  • HAN Guoquan ,
  • YU Bing ,
  • CHEN Daiwen ,
  • XIANG Zhentian ,
  • CHEN Yu ,
  • CHEN Hong ,
  • MAO Qian
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  • 1. Institue of Animal Nutrition, Sichuan Agricultural University, Ya'an 625014, China;
    2. Key Laboratory of Animal Disease-Resistant Nutrition of China Ministry of Education, Sichuan Agricultural University, Ya'an 625014, China

Received date: 2011-09-27

  Online published: 2012-02-28

Supported by

教育部长江学者和创新团队发展计划项目资助(IRTO555);现代农业产业技术体系建设专项资金资助(CARS-36);博士后资金资助(04310610)

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摘要

本研究旨在探讨苏氨酸对体外培养感染伪狂犬病毒(PRV)猪空肠上皮细胞免疫相关基因表达的影响。试验选用IPEC-J2细胞为细胞模型,分为4个处理,对照组为未经PRV感染、苏氨酸浓度53.45 mg/L,Ⅰ、Ⅱ、Ⅲ组为试验组,均经PRV感染,苏氨酸浓度分别为53.45、106.90、160.35 mg/L,每组3个重复,每个重复4孔。37 ℃、5% CO2培养后1、6、12、24 h每组取1个重复采集细胞,用实时荧光定量PCR方法检测白介素1β(IL-1β)、白介素6(IL-6)、白介素15(IL-15)、肿瘤坏死因子α(TNF-α)及转化生长因子β1(TGF-β1)mRNA表达量。结果表明:1)PRV感染极显著降低了IL-1β mRNA表达量(P=0.001),苏氨酸水平(P=0.830)、时间与苏氨酸水平交互作用(P=0.249)的影响均不显著;2)PRV感染对IL-6 mRNA表达量影响不显著(P=0.162),随苏氨酸水平升高,IL-6 mRNA表达量极显著地先升高后降低(P=0.002),时间与苏氨酸水平交互作用的影响显著(P=0.012);3)PRV感染极显著提高了IL-15 mRNA表达量(P=0.000),提高苏氨酸水平极显著提高了IL-15 mRNA表达量(P=0.000),时间与苏氨酸水平交互作用的影响亦极显著(P=0.000);4)PRV感染极显著提高了TNF-α mRNA表达量(P=0.000),苏氨酸水平(P=0.113)、时间与苏氨酸水平交互作用(P=0.195)的影响均不显著;5)PRV感染极显著提高了TGF-β1 mRNA表达量(P=0.000),时间与苏氨酸水平交互作用的影响显著(P=0.015),苏氨酸水平的影响不显著(P=0.055)。结果提示,补充苏氨酸对感染PRV的IPEC-J2细胞先天性免疫功能具有分子表达水平的调控作用,总体上能够抑制IL-1β、TNF-α、TGF-β1基因表达,加强IL-6、IL-15 基因表达,但影响具有时间效应。

关键词： 苏氨酸; 猪空肠上皮细胞; 伪狂犬病毒; 免疫相关基因

本文引用格式

韩国全 , 余冰 , 陈代文 , 相振田 , 陈渝 , 陈洪 , 毛倩 . 苏氨酸对体外培养感染伪狂犬病毒猪空肠上皮细胞免疫相关基因表达的影响[J]. 动物营养学报, 2012 , 24(3) : 487 -496 . DOI: 10.3969/j.issn.1006-267x.2012.03.015

Abstract

The present study was conducted to evaluate the effects of threonine (Thr) on expression of immune-related genes of porcine intestinal epithelial cell infected with pseudorabies virus (PRV) in vitro. IPEC-J2 cells were selected as the cell model, and were divided into four treatments. Cells in the control group were un-infected with PRV and cultured with Thr at the concentration of 53.45 mg/L in culture medium, and cells in experimental groups (groups Ⅰ, Ⅱ and Ⅲ) were infected with PRV and cultured with Thr at concentrations of 53.45, 106.90 and 160.35 mg/L in culture medium, respectively. There were 3 replicates in each group and 4 wells per replicate. After cultured in 37 ℃ and with 5% CO2 for 1, 6, 12 and 24 h, cells in one replicate from each group were collected, respectively. Then mRNA expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-15 (IL-15), tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) in those cells were determined by real-time PCR. The results showed as follows: 1) IL-1β mRNA expression level was significantly reduced by PRV infection (P=0.001), however effects of Thr level (P=0.830) and its interaction with time (P=0.249) were not significant; 2) IL-6 mRNA expression level was not significantly reduced by PRV infection (P=0.162), while was significantly increased at first and then decreased with the increasing of Thr level (P=0.002), and the interaction between time and Thr level was significant (P=0.012); 3) PRV infection significantly increased the mRNA expression of IL-15 (P=0.000), and the expression level was significantly increased with the increasing of Thr level (P=0.000), the interaction between time and Thr level was also significant (P=0.000); 4) PRV infection significantly increased the mRNA expression level of TNF-α (P=0.000), however effects of Thr level (P=0.113) and its interaction with time (P=0.195) were not significant; 5) PRV infection significantly increased the mRNA expression level of TGF-β1 (P=0.000), and the interaction between Thr level and time was significant (P=0.015), but the effect of Thr level was not significant (P=0.055). The results indicate that Thr supplementation can regulate the innate immunity function of IPEC-J2 cells infected with PRV at molecular level, and can generally inhibit gene expression of IL-1β, TNF-α and TGF-β1, but enhance gene expression of IL-6 and IL-15, while the effects vary with the time.

Key words： threonine; porcine jejunal epithelial cell; pseudorabies virus; immune-related genes

参考文献

[1] ROSE W C.Feeding experiments with mixtures of highly purified amino acids.Ⅰ.The inadequacy of diets containing nineteen amino acids[J].The Journal of Biological Chemistry,1931,94:155-165.
[2] MCCOY R H,MEYER C E,ROSE W C.Feeding experiments with mixtures of highly purified amino acids.Ⅷ.Isolation and identification of a new essential amino acid[J].The Journal of Biological Chemistry,1935,112:283-302.
[3] SIMONI R D,HILL R L,VAUGHAN M.The discovery of the amino acid threonine:the work of William C.Rose[J].The Journal of Biological Chemistry,2002,277(37):E25.
[4] FULLER M F,MILNE A,HARRIS C I,et al.Amino acid losses in ileostomy fluid on a protein-free diet[J].The American Journal of Clinical Nutrition,1994,59(1):70-73.
[5] STOLL B,HENRY J,REEDS P J,et al.Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets[J].The Journal of Nutrition,1998,128(3):606-614.
[6] WANG X,QIAO S Y,LIU M,et al.Effects of graded levels of true ileal digestible threonine on performance,serum parameters and immune function of 10-25 kg pigs[J].Animal Feed Science and Technology,2006,129(3/4):264-278.
[7] FAURE M,CHONE F,METTRAUX C,et al.Threonine utilization for synthesis of acute phase proteins,intestinal proteins,and mucins is increased during sepsis in rats[J].The Journal of Nutrition,2007,137(7):1802-1807.
[8] REMOND D,BUFFIERE C,GODIN J P,et al.Intestinal inflammation increases gastrointestinal threonine uptake and mucin synthesis in enterally fed minipigs[J].The Journal of Nutrition,2009,139(4):720-726.  
[9] SCHIERACK P,NORDHOFF M,POLLMANN M,et al.Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine[J].Histochemistry and Cell Biology,2006,125(3):293-305.  
[10] NARITA M,KUBO M,FUKUSHO A,et al.Necrotizing enteritis in piglets associated with Aujeszky's disease virus infection[J].Veterinary Pathology,1984,21(4):450-452.
[11] 韩国全.含沙门菌内源启动子新型双功能表达载体的构建及其在猪瘟病毒疫苗研究中的应用 .博士学位论文.雅安:四川农业大学,2009.
[12] REED L J,MUENCH H.A simple method of estimating fifty percent endpoints[J].American Journal of Hygiene,1938,27(3):493-497.
[13] PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45.
[14] MONCADA D M,KAMMANADIMINTI S J,CHADEE K.Mucin and Toll-like receptors in host defense against intestinal parasites[J].Trends in Parasitology,2003,19(7):305-311.  
[15] BERSCHNEIDER H M.Development of normal cultured small intestinal epithelial cell lines which transport Na and Cl[J].Gastroenterology,1989,96:A41.
[16] KAEFFER B,BOTTREAU E,VELGE P,et al.Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid[J].European Journal of Cell Biology,1993,62(1):152-162.
[17] LEONARD R P,SPEER V C.Threonine requirement for reproduction in swine[J].Journal of Animal Science,1983,56(6):1345-1353.
[18] WANG X,QIAO S,YIN Y,et al.A deficiency or excess of dietary threonine reduces protein synthesis in jejunum and skeletal muscle of young pigs[J].The Journal of Nutrition,2007,137(6):1442-1446.
[19] CASTELLANOS M,CASTILLO J,GARCIA M M,et al.Inflammation-mediated damage in progressing lacunar infarctions:a potential therapeutic target[J].Stroke,2002,33(4):982-987.  
[20] VAN SNICK J.Interleukin-6:an overview[J].Annual Review of Immunology,1990,8:253-278.
[21] RADDATZ D,BOCKEMUHL M,RAMADORI G.Quantitative measurement of cytokine mRNA in inflammatory bowel disease:relation to clinical and endoscopic activity and outcome[J].European Journal of Gastroenterology & Hepatology,2005,17(5):547-557.  
[22] TURKAY C,KASAPOGLU B.Noninvasive methods in evaluation of inflammatory bowel disease:where do we stand now? An update[J].Clinics (Sao Paulo),2010,65(2):221-231.  
[23] HIROMATSU T,YAJIMA T,MATSUGUCHI T,et al.Overexpression of interleukin-15 protects against Escherichia coli-induced shock accompanied by inhibition of tumor necrosis factor-alpha-induced apoptosis[J].Journal of Infectious Diseases,2003,187(9):1442-1451.  
[24] YAJIMA T,NISHIMURA H,ISHIMITSU R,et al.Overexpression of IL-15 in vivo increases antigen-driven memory CD8+ T cells following a microbe exposure[J].The Journal of Immunology,2002,168(3):1198-1203.
[25] RODELLA L,ZAMAI L,REZZANI R,et al.Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells[J].British Journal of Haematology,2001,115(2):442-450.  
[26] GRABSTEIN K H,EISENMAN J,SHANEBECK K,et al.Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor[J].Science,1994,264(5161):965-968.  
[27] ASHKAR A A,BLACK G P,WEI Q,et al.Assessment of requirements for IL-15 and IFN regulatory factors in uterine NK cell differentiation and function during pregnancy[J].The Journal of Immunology,2003,171(6):2937-2944.
[28] ASHKAR A A,ROSENTHAL K L.Interleukin-15 and natural killer and NKT cells play a critical role in innate protection against genital herpes simplex virus type 2 infection[J].Journal of Virology,2003,77(18):10168-10171.  
[29] KAPADIA S,LEE J,TORRE-AMIONE G,et al.Tumor necrosis factor-alpha gene and protein expression in adult feline myocardium after endotoxin administration[J].Journal of Clinical Investigation,1995,96(2):1042-1052.  
[30] SU C,SALZBERG B A,LEWIS J D,et al.Efficacy of anti-tumor necrosis factor therapy in patients with ulcerative colitis[J].The American Journal of Gastroenterology,2002,97(10):2577-2584.  
[31] MOSES H L,YANG E Y,PIETENPOL J A.TGF-beta stimulation and inhibition of cell proliferation:new mechanistic insights[J].Cell,1990,63(2):245-247.  
[32] MOSES H L,ARTEAGA C L,ALEXANDROW M G,et al.TGF beta regulation of cell proliferation[J].Princess Takamatsu Symp,1994,24:250-263.
[33] BAI L,YU Z,WANG C,et al.Dual role of TGF-beta1 on Fas-induced apoptosis in lung epithelial cells[J].Respiratory Physiology & Neurobiology,2011,177(3):241-246.  
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