绵羊Toll样受体家庭在肺泡巨噬细胞的分布及脂多糖(LPS)刺激对TLR2、TLR4表达的影响

doi:10.7668/hbnxb.2010.01.006

华北农学报 ›› 2010, Vol. 25 ›› Issue (1): 30-35. doi: 10.7668/hbnxb.2010.01.006

所属专题： 畜牧；

绵羊Toll样受体家庭在肺泡巨噬细胞的分布及脂多糖(LPS)刺激对TLR2、TLR4表达的影响

盛金良¹, 陈创夫¹, 杨霞¹, 王远志², 张辉¹

1. 新疆石河子大学,动物科技学院, 新疆石河子 832003;
2. 新疆石河子大学,医学院, 新疆石河子 832003

收稿日期:2009-12-24 出版日期:2010-02-28
通讯作者: 陈创夫(1962-),男,广东人,教授,博士生导师,博士,主要从事家畜病理学和免疫遗传和抗病机理研究.
作者简介:盛金良(1974-),男,新疆石河子人,副教授,博士,主要从事家畜病理学和免疫遗传和抗病机理研究.
基金资助:
国际科技合作项目资助(2006DFA33740);国家自然科学基金项目资助(30960276)

Distribution of Toll-like Receptors(TLRs) and TLR2,TLR4 Expression During LPS Stimulus Sheep Alveolar Macrophage

SHENG Jin-liang1¹, CHEN Chuang-fu1¹, YANG Xia1¹, WANG Yuan-zhi2², ZHANG Hui1¹

1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China;
2. Shihezi School of Medicine, Shihezi University, Shihezi 832003, China

Received:2009-12-24 Published:2010-02-28

摘要/Abstract

摘要： 为确定toll样受体家族在绵羊肺泡巨噬细胞的分布及脂多糖(LPS)刺激过程中TLR2、4的动态表达规律.通过RT-PCR方法检测绵羊TLR1-TLR10表达分布;以α-tubulin和GAPDH为内标基因,对绵羊TLR2、4进行克隆和测序,将重组质粒作为标准品建立实时定量PCR标准曲线,通过实时定量PCR方法检测LPS诱导0～48 h TLR2、4的动态表达.结果表明,在绵羊肺泡巨噬细胞中检测到TLR1、2、4、、7、8、9、10的表达,而TLR3、5未见表达;α-tubulin和GAPDH在LPS诱导前后表达量存在显著差异,均不适合作为内标基因,以使用的细胞数和总RNA量进行均一化校正,发现TLR2、4在诱导后20 min就达到高峰,且在整个诱导过程中维持较高水平.绵羊肺泡巨噬细胞TLR2、4对LPS刺激的应答反应很灵敏,参与机体的早期应答反应.

关键词: 绵羊, 肺泡巨噬细胞, 脂多糖, toll样受体, 实时定量PCR

Abstract: 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.

Key words: Sheep, Alveolar macrophage, LPS, Toll like receptor, Real-time PCR

中图分类号:

S852.4+2

盛金良, 陈创夫, 杨霞, 王远志, 张辉. 绵羊Toll样受体家庭在肺泡巨噬细胞的分布及脂多糖(LPS)刺激对TLR2、TLR4表达的影响[J]. 华北农学报, 2010, 25(1): 30-35. doi: 10.7668/hbnxb.2010.01.006.

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.

http://www.hbnxb.net/CN/Y2010/V25/I1/30

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绵羊Toll样受体家庭在肺泡巨噬细胞的分布及脂多糖(LPS)刺激对TLR2、TLR4表达的影响

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绵羊Toll样受体家庭在肺泡巨噬细胞的分布及脂多糖(LPS)刺激对TLR2、TLR4表达的影响

Distribution of Toll-like Receptors(TLRs) and TLR2,TLR4 Expression During LPS Stimulus Sheep Alveolar Macrophage

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