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.
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
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.
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