Zhang Jin-xiang, Wang Hui, Wu He-shui, Jiang Chun-fang, Zheng Qi-chang
Department of Emergency Surgery, Union Hospital Affiliated to Huazhong University of Sciences and Technology, Wuhan 430022, China.
Zhonghua Yi Xue Za Zhi. 2006 May 23;86(19):1323-6.
To construct a eukaryotic expression vector carrying the small hairpin RNA (shRNA) for Toll-like receptor 4 (TLR4) mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by rat RAW264.7 macrophages induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism.
The H1 promotor and double BbsIrestrict endoenzyme site from the plasmid psiRNA-hH1neo were cloned into the reporter gene plasmid pEGFP-C1 at the MluIrestrict endoenzymic site, thus forming the plasmid pEGFP-H1/siRNA containing Bbs site and reporter EGFP gene. Then an oligo nuclear hairpin sequence targeting TLR4 gene was designed by the internet tool siRNA Wizard and then inserted into the plasmid pEGFP-H1/siRNA so as to form the plasmid pEGFP-H1/TLR4-siRNA. Rat macrophages of the line RAW264.7 were cultured and transfected with pEGFP-H1/TLR4-siRNA mediated by lipofectamine 2000. Another RAW264.7 cells were transfected with pEGFP-H1/control sequence-siRNA or blank plasmid. Lipopolysaccharide was added into the 3 kinds of culture fluid for 2 and 68 hours respectively. ELSA was used to detect the levels of tumor necrosis factor-alpha (TNF-alpha) in the supernatants.
Restriction endonuclease analysis showed that the construction pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene was successful. The expression of EGFP gene was 50% +/- 8%. The TNF-alpha level of the TLR4-siRNA transfection group 2 hours and after transfection was 825 pg/ml +/- 136 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (2190 pg/ml +/- 359 pg/ml and 1265 pg/ml +/- 283 pg/ml respectively, both P < 0.01). The TNF-alpha level of the TLR4-siRNA transfection group 8 hours and after transfection was 1179 pg/ml +/- 240 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (4720 pg/ml +/- 227 pg/ml and 4689 pg/ml +/- 310 pg/ml respectively, both P < 0.01).
shRNA targeting TLR4 gene can inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.
构建携带Toll样受体4（TLR4）mRNA的小发夹RNA（shRNA）及增强型绿色荧光蛋白（EGFP）报告基因的真核表达载体，并通过RNA干扰机制转染及表达靶向TLR4基因的shRNA，研究其对脂多糖（LPS）刺激诱导的大鼠RAW264.7巨噬细胞释放细胞因子的抑制作用。
将质粒psiRNA-hH1neo中的H1启动子和双BbsI限制性内切酶位点克隆到报告基因质粒pEGFP-C1的MluI限制性内切酶位点，从而形成含有Bbs位点和报告基因EGFP的质粒pEGFP-H1/siRNA。然后通过网络工具siRNA Wizard设计靶向TLR4基因的寡核发夹序列，并将其插入质粒pEGFP-H1/siRNA中，形成质粒pEGFP-H1/TLR4-siRNA。培养大鼠RAW264.7巨噬细胞，并用脂质体2000介导转染pEGFP-H1/TLR4-siRNA。另一组RAW264.7细胞转染pEGFP-H1/对照序列-siRNA或空白质粒。分别向3种培养液中加入脂多糖2小时和68小时。采用酶联免疫吸附测定（ELSA）检测上清液中肿瘤坏死因子-α（TNF-α）的水平。
限制性内切酶分析表明，携带TLR4基因发夹RNA和报告基因EGFP的pEGFP-H1/TLR4-siRNA构建成功。EGFP基因的表达率为50%±8%。TLR4-siRNA转染组转染后2小时TNF-α水平为825 pg/ml±136 pg/ml，显著低于pEGFP-H1/对照序列-siRNA组和空白质粒组（分别为2190 pg/ml±359 pg/ml和1265 pg/ml±283 pg/ml，均P<0.01）。TLR4-siRNA转染组转染后8小时TNF-α水平为1179 pg/ml±240 pg/ml，显著低于pEGFP-H1/对照序列-siRNA组和空白质粒组（分别为4720 pg/ml±227 pg/ml和4689 pg/ml±310 pg/ml，均P<0.01）。
靶向TLR4基因的shRNA可抑制LPS诱导的RAW264.7细胞释放TNF-α。
