To determine the distribution of toll-like receptor family(TLRs) in sheep alveolar macrophage(AM)and the expression pattern of TLR2,4 during lipopolysaccharide(LPS) stimulation.Reverse transcription PCR(RT-PCR) was used to detect the distribution of sheep TLR1-TLR10 gene expression.α-tubulin and GAPDH were testified as valid house keeping genes(HKG).Real-time PCR standard curve was established with sheep TLR2,4 genes recombinant plasmid as a standard criteria,dynamic expression of sheep TLR2,4 in AM with LPS-induced 0-48 h were detected.TLR1,2,4,6,7,8,9,10 genes expression were detected in sheep alveolar macrophages except that of TLR3,5.α-tubulin and GAPDH have significant increase in LPS post stimulation and were not suitable as HKG.The specimen was normalized with cell number and total RNA.TLR2,4 mRNA expression rapidly increased post-stimulation and peaked at 20 min post-stimulation and this high level was maintained throughout the procedure.Sheep TLR2,4 were sensitive in LPS-stimulated AM and participate in early stage of immune response.
SHENG Jin-liang1
,
CHEN Chuang-fu1
,
YANG Xia1
,
WANG Yuan-zhi2
,
ZHANG Hui1
. Distribution of Toll-like Receptors(TLRs) and TLR2,TLR4 Expression During LPS Stimulus Sheep Alveolar Macrophage[J]. Acta Agriculturae Boreali-Sinica, 2010
, 25(1)
: 30
-35
.
DOI: 10.7668/hbnxb.2010.01.006
[1] Menzies M,Ingham A.Identification and expression of Toll-like receptors 1-10 in selected bovine and ovine tissues[J].Veterinary Immunology and Immunopathology,2006,109:23-30.
[2] Aderem A,Ulevitch R J.Toll-like receptors in the induction of the innate immune response[J].Nature,2000,406:782-787.
[3] Cario E,Rosenberg I M,Brandwein S L,et al.Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing toll-like receptors[J].J Immunol,2000,164:966-972.
[4] Matsuguchi T,Musikachroen T,Ogawa T,et al.Gene expressions of toll-like receptors 2,but not toll-like receptor 4,is induced by LPS and inflammatory cytokines in mouse macrophages[J].J Immunol,2000,165:5767-5772.
[5] Matsuguchi T,Takagi K,Musikacharen T.Gene expressions of lipopolysaccharide receptors,toll-like receptors 2 and 4,are differently regulated in mouse T lymphocytes[J].Blood,2000,95:1378-1385.
[6] Faure E,Thomas L,Xu H,et al.Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor2 and Toll-like receptor4 expression in human endothelial cells:role of NF-kappaB activation[J].J Immunol,2001,166:2018-2024.
[7] Muzio M,Bosisio D,Polentarutti N,et al.Differential expression and regulation of toll-like receptors(TLR) in human leukocytes:selective expression of TLR3 in dendritic cells[J].J Immunol,2000,164:5998-6004.
[8] Sabroe I,Jones E C,Usher L R,et al.Toll-like receptor(TLR)2 and TLR4 in human peripheral blood granulocytes:a critical role for monocytes in leukocyte lipoplysaccharide responses[J].J Immunol,2002,168:4701-4710.
[9] Akira S.Toll-like receptors and innate immunity[J].Adv Immunol,2001,78:1-56.
[10] Yakobson B,Brenner J,Ungar-waron H,et al.Cellular immune response cytokine expression during the initial stage of bovine leukemia virus(BLV) infection determines the disease progression to persistent lymphocytosis[J].Comp Immunol,Microbiol Infect Dis,2000,23:197-208.
[11] Leutenegger C M,Alluwaimi A M,Smith W L,et al.Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction[J].Vet Immunol Immunopathol,2000,77:275-287.
[12] 盛金良,陈创夫,李有文,等.绵羊肺泡巨噬细胞的分离、培养和形态学观察[J].石河子大学学报,2007,1(25):41-45.
[13] Chang J S,Russell G C,Jann O,et al.Molercular cloning and characterization of toll like receptors 1-10 in sheep[J].Veterinary Immunology and Immunopahtology,2009,15,127(1-2):94-105.
[14] Giulietti A,Overbergh L,Valckx D,et al.An overview of real-time quantitative PCR:applications to quantify cytokine gene expression[J].Methods,2001,25:386-391.
[15] Medvedev A E,Kopydlowski K M,Vogel S N,et al.Inhibition of lipopolysaccharide-induced signal transduction in endotoxin-tolerized mouse macrophages:dysregulation of cytokine,chemokine,and toll-like receptor2 and 4 gene expression[J].J Immunol,2000,164:5564-5574.
