TLR4基因的shRNA对内毒素刺激下RAW264．7细胞TLR2表达的影响及机制  

Effects of short hairpin RNA targeting TLR4 on TLR2 expression induced by lipopolysaecharide in murine macrophage cell lime RAW 264.7

作　　者：徐建波[1] 吴河水[1] 张进祥[2] 王琳[3] 陈凤花[4] 王春友[1] 

机构地区：[1]华中科技大学附属协和医院胰腺外科,武汉430022 [2]华中科技大学附属协和医院急诊外科,武汉430022 [3]华中科技大学附属协和医院儿科,武汉430022 [4]华中科技大学附属协和医院检验科,武汉430022

出　　处：《中华急诊医学杂志》2008年第3期267-270,共4页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金资助项目（30200722）

摘　　要：目的观察针对T0u样受体4的发夹结构RNA（short hairpin RNA，shRNA）对内毒素刺激后小鼠巨噬细胞系RAW264．7细胞Toll样受体2表达的影响，并探讨其机制。方法将携带增强型绿色荧光蛋白（EGFP）及shRNA克隆位点的质粒pEGFP／TLR4-siRNA，运用脂质体Lipofectamine2000转染至小鼠巨噬细胞系RAW264．7。转染24h后予新霉素（G418）筛选获取稳定转染细胞株。然后将细胞分为三组：空白组（Blank group），未转染细胞LPS刺激组（LPS group）和稳定转染细胞LPS刺激组（LPS-TLR4-siRNA group），采用荧光定量PCR及流式细胞学方法检测各组细胞TLR2 mRNA和蛋白的表达，Westem blot检测NF—κB p65的表达，同时用ELISA方法检测细胞分泌的TNF-α水平的变化。结果将pEGFP／TLR4-siRNA转染至RAW264．7细胞后，增强型荧光蛋白的表达为（45．25±9．23）％。LPS-TLR4-siRNA组细胞的TLR2mRNA及蛋白的表达均明显低于LPS组（P〈0．05），同时NF-κBp65表达下调及分泌TNF-α的水平下降（P〈0．01）。结论针对TLR4 mRNA的shRNA能降低LPS刺激下的RAW264．7细胞TLR2的表达。Objective To study the expression of Toll-like receptor 2 following short hairpin RNAi （shRNA） targeting Toll-like receptor-4 （TLR4） under the milieu of LPS stimtdation in macrophage cell line RAW 264.7 cells in order to explore its mechanism. Method The plasmid of pEGFP/TLR4-shRNA were transfected into RAW264,7 cells. The efficiency of transfection was detected by observing the expression of EGFP （Enhanced Green Fluorescent Protein, EGFP）. Cells after 24 hour of transfecfion were given the pressure of selective medium （G418） to get stable cell lines. Cells were divided into three groups： Blank group, LPS group, LPS-TLR4-shRNA group. The synthesis of TLR2 protein was determined by flow cytometric analysis （FCM） and their gene expression were analyzed by reverse tmnseripfion polymerase chain reaction （RT-PCR）. The variation of NF-κB p65 expression in three groups was analyzed by western blot. TNF-α production of the supernatants was measured with the ELISA （Enzyme-Linked Immunosorbent Assay） following the protocol presented by manufacturer. Results The expression of EGFP gene was （45.25 ± 9.23） % after transfection of the plasmid pEGFP/TLR4-sh RNA. The expression of mRNA and protein in LPS-TLR4-shRNA group decreased compared with that in LPS group. NF-κB in LPS-TLR4-shRNA group decreased compared with that in LPS group, as well as the level of TNF-α in the supernatants. Conclusions Short hairpin RNA （shRNA） targeting TLR4 gene significantly inhibit the expression of TLR2.

关 键 词：RNA干扰 内毒素 巨噬细胞 TOLL样受体 

分 类 号：R285.5[医药卫生—中药学]