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Construction of Toll-like receptor 4 siRNA expression vector and its inhibition effect on expression of TLR4 in RAW264.7 cells in vitro
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 24
- DOI:
- 10.3321/j.issn:1000-2790.2007.24.001
- Key Word:
- Toll样受体4;RNA,小分子干扰;脂多糖类;巨噬细胞
Abstract： 目的:构建针对小鼠Toll样受体4(TLR4)的小干扰RNA(siRNA)表达载体,体外评价对RAW264.7细胞TLR4 mRNA及蛋白水平的抑制效果;观察对TNF-α,MIP-2水平及p38-MAPK,ERK1/2磷酸化水平的影响.方法:根据siRNA设计原则并经BLAST比对,设计合成7条siRNA双链复合体,体外退火后在T4连接酶作用下连接入重组载体pSilencerTM4.1-CMV neo plasmid,连接产物转化JM 109感受态细胞,筛选阳性克隆并提取重组体,行Hind Ⅲ及Bam H I双酶切,酶切产物经聚丙稀酰胺凝胶电泳确定插入片段,测序鉴定;RT-PCR检测TLR4 mRNA水平变化,间接免疫荧光及Western Blot检测TLR4蛋白水平变化;用LPS刺激转染细胞,观察TN-α及MIP-2水平的变化,Western Blot检测p38-MAPK及ERK1/2磷酸化水平的变化.结果:双酶切及测序证实重组载体构建成功;体外实验表明,RAW264.7细胞中TLR4 mRNA及蛋白水平均降低;TLR4 siRNA抑制LPS对TNF-α及MIP-2表达的上调作用,LPS对p38-MAPK及ERK1/2的活化作用亦被TLR4 siRNA下调.结论:TLR4 siRNA表达载体在体外可下调RAW264.7细胞的TLR4表达,抑制丝裂原活化蛋白激酶(MAPK)信号通路,对LPS诱导的RAW264.7细胞TNF-α及MIP-2的分泌有抑制作用,TLR4 siRNA可望作为调控LPS信号通路的一个有效工具.
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