IRAK-M短发夹RNA对RAW264.7细胞内毒素耐受性的抑制作用  

Inhibitory effect of short hairpin RNA targeting IRAK-M gene for endotoxin tolerance in RAW264.7 cells

作　　者：李旭宏[1] 陈先锋[2] 游海波[2] 刘海忠[2] 刘作金[2] 龚建平[2] 

机构地区：[1]重庆三峡中心医院肝胆外科,重庆市404000 [2]重庆医科大学附属第二医院肝胆外科,重庆市400010

出　　处：《世界华人消化杂志》2009年第5期444-448,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30471696;No.30500473~~

摘　　要：目的:观察白介素-1受体相关激酶-M(IRAK-M)基因沉寂后RAW264.7细胞内毒素耐受性的改变,进而探讨IRAK-M在内毒素耐受形成中的作用.方法:构建表达IRAK-M短发夹RNA(shRNA)的阳性重组质粒(pshIRAK-M-A)和阴性重组质粒(pshIRAK-M-B),转染RAW264.7(小鼠单核巨噬细胞系)细胞;各组细胞经10μg/L脂多糖(LPS)预处理24h后(或直接)用100μg/LLPS刺激;3h后,酶联免疫吸附法(ELISA)检测培养液中TNF-α水平,逆转录聚合酶链反应(RT-PCR)检测细胞中的TNF-α mRNA表达水平,蛋白印迹法(Western blotting)检测细胞中IRAK-M蛋白的表达,凝胶电迁移率分析法(EMSA)检测细胞中NF-κB的活性.结果:pshIRAK-M-A对IRAK-M蛋白表达抑制率为83%左右,pshIRAK-M-B对IRAK-M蛋白表达无明显抑制作用;两种转染细胞在用100μg/LLPS直接刺激后,其培养液中TNF-α水平、细胞内TNF-α mRNA表达及NF-κB活性无显著性差异;两种转染细胞对LPS的再次应答均明显弱于初次应答细胞(P<0.05),但pshIRAK-M-A转染组细胞对LPS的再次应答明显强于pshIR AK-M-B转染组细胞(P<0.05).结论:IRAK-M表达受抑导致细胞内毒素耐受性减弱,IRAK-M在内毒素耐受形成中起重要作用.AIM： To explore the role of IRAK-M in the development of endotoxin tolerance by silencing the gene of interleukin-1 receptor associated kinase-M （IRAK-M） in RAW264.7 cells by the specific short hairpin RNA （shRNA）. METHODS： The recombinant plasmids expressing effective shRNA （pshIRAK-M-A） or invalidated shRNA （pshIRAK-M-B） targeting IRAK-M gene were constructed and were transfected into RAW264.7 cells. Cells were stimulated by 100 μg/L LPS with or without 10 μg/L LPS pretreatment. At 3 h after LPS stimulation, the TNF-R level in culture medium was measured using ELISA, the expression of TNF-α mRNA was detected using RT-PCR, the expression of IRAK-M protein was detected by Western blotting and the activation of NF-κB was detected by electrophoretic mobility shift assay （EMSA）. RESULTS： The gene expression of IRAK-M in RAW264.7 cells was reduced about 83% by pshI- RAK-M-A transfection, pshIRAK-M-B transfection hardly inhibited IRAK-M gene expression. At 3 h after 100 μg/L LPS stimulation, the TNF-α level in culture medium, the TNF-α mRNA expression and the NF-κB activity in cells had no significant difference between two kinds of transfected cells without LPS pretreatment. Two kinds of transfected cells both showed obviously attenuated response to 100 μg/L LPS stimulation after LPS pretreatment （P 〈 0.05）, but this response was more intense in cells transfected by pshIRAK-M-A than in cells transfected by pshIRAK-M-B （P 〈 0.05） CONCLUSION： The inhibition of IRAK-M gene expression results in the attenuation of endotoxin tolerance in RAW264.7 cells, and IRAK-M plays an important role in the development of endotoxin tolerance.

关 键 词：RAW264.7细胞 内毒素耐受 白介素-1受体相关激酶-M 凝胶电迁移率分析法 

分 类 号：R363[医药卫生—病理学] R927.12[医药卫生—基础医学]