针对Toll样受体4基因的小发夹结构RNA对RAW264·7细胞炎症因子分泌的抑制作用

Inhibition of rat RAW264.7 macrophage inflammatory cytokines release by small hairpin RNAi targeting Toll-like receptor

目的构建针对Toll样受体(TLR)4mRNA的小发夹结构RNA(shRNA)真核表达载体pEGFP-siRNA/TLR4,检测该shRNA诱导的RNA干扰(RNAi)对脂多糖刺激RAW264·7细胞分泌炎症因子的抑制作用,以探讨针对TLR4基因的RNAi对RAW264·7细胞炎症反应的抑制作用。方法构建携带增强型绿色荧光蛋白(EGFP)及shRNA克隆位点的质粒pEGFP-H1/siRNA,运用网络工具siRNAWizard(http://www·sirnawizard·com/)设计针对TLR4mRNA的寡核苷酸片段。将其克隆入pEGFP-H1/siRNA,构建表达EGFP及TLR4-shRNA的真核表达质粒pEGFP-H1/TLR4-siRNA。运用脂质体转染技术将pEGFP-H1/TLR4-siRNA转染至培养的小鼠巨噬细胞系RAW264·7,观察细胞系中荧光蛋白的表达强度;再运用脂多糖刺激转染的RAW264·7细胞,运用ELISA方法检测该细胞系分泌炎症因子水平的变化。结果质粒pEGFP-H1/siRNA及pEGFP-H1/TLR4-siRNA分别用BbsⅠ和MluⅠ酶切电泳后,前者出现4·9kb和340bp的的条带,后者出现5·0kb和220bp的条带,与实验设计的质粒长度一致,且测序证实克隆序列正确。将pEGFP-H1/TLR4-siRNA转染至RAW264·7细胞后,EGFP的表达率为50%±8%。用脂多糖刺激后,TLR4-SiRNA转染组细胞培养液TNF-α水平(2h:825pg/ml±136pg/ml;8h:2190pg/ml±359pg/ml)明显低于对照siRNA转染组(2h:1179pg/ml±240pg/ml;8h:4720pg/ml±227pg/ml,均P<0·01)。结论针对TLR4mRNA的shRNA可能通过RNAi机制对脂多糖诱导的RAW264·7细胞炎症因子的分泌有明显的抑制作用。

Objective 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 lipopolysaecharide （LPS） stimulation through transfecfion and expression of shRNA targeting TLR4 gene via the RNAi mechanism. Methods The H1 promotur and double Bbs I restrict endoenzyme site from the plasmid psiRNA-hHlnec were cloned into the reporter gene plasmid pEGFP-C1 at the Mlu I restrict 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 intemet tool siRNA Wizard and then inserted into the plasmid pEGFPoH1/siRNA so as to form the plasmid pEGFPoH1/TLR4-siRNA. Rat macmphages 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-α） in the supernatants. Results Restriction endonuclease analysis showed that the construction pEGFP-H1/TLR4siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene was successful. The expression of EGFP gene was 50% ± 8% . The TNF-α level of the TLR4-siRNA transfeetion group 2 hours and after transfection was 825 pg/ml ±136 pg/ml, significantly lower than those of the pEGFP-H1/eontrol sequencesiRNA and blank plasmid groups （2190 pg/ml ±359 pg/ml and 1265 pg/ml ± 283 pg/ml respectively, both P〈0.01）. The TNF-α 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/eontrol sequenee-siRNA and blank plasmid groups （4720 pg/ml ± 227 pg/ml and 4689 pg/ml± 310 pg/ml respectively, both P〈0.01 ）. Conclusion shRNA targeting TLR4 gene can inhibit the TNF-α release by RAW264.7 cells evoked by LPS.