TLR4基因的沉默下调了NOD1信号通路在RAW264．7细胞中抗烟曲霉孢子刺激作用  

Silencing TLR4 Down-regulates NOD1-mediated Inflammatory Responses by Aspergillus fumigatus Conidia Stimulation in RAW264.7 Macrophages

作　　者：饶振华[1] 谢卫华[2] 苏明声[1] 龙凯[2] 罗闳丹[1] 谢小梅[1] 

机构地区：[1]江西中医学院现代中药制剂教育部重点实验室,南昌330004 [2]江西中医学院药学院,南昌330004

出　　处：《中国科学：生命科学》2012年第6期503-512,共10页Scientia Sinica(Vitae)

基　　金：国家自然科学基金(批准号:30560147和30760236)资助项目

摘　　要：为研究单核巨噬细胞RAW264.7受烟曲霉孢子刺激时NOD1和TLR4信号通路在细胞处于正常状态和免疫抑制状态时发挥的作用,以及沉默TLR4基因后对NOD1信号通路的影响,通过建立细胞免疫抑制模型,利用RNAi技术将TLR4-siRNA转染细胞24h后给予烟曲霉孢子刺激12h,运用RT-PCR和Western blot法检测细胞受烟曲霉孢子刺激后NOD1,TLR4,RIP2,MyD88 mRNA及TNF-α蛋白表达变化.研究发现:(1)给予RAW264.7细胞含免疫抑制剂甲泼尼龙琥珀酸钠浓度为0.04mg/mL的RPMI1640培养液培养12h后,细胞抑制效果最佳,存活率约为90%.(2)正常RAW264.7细胞受烟曲霉孢子刺激时,NOD1和TLR4信号通路被激活,通过释放促炎细胞因子TNF-α发挥抗烟曲霉孢子刺激作用,当细胞处于免疫抑制状态时,NOD1和TLR4信号通路不能激发其有效的免疫应答.(3)TLR4-siRNA转染RAW264.7细胞,沉默效率达83%.(4)TLR4基因的沉默,使NOD1信号通路不能被很好地激活来抵抗烟曲霉孢子对细胞的刺激作用,表明沉默TLR4基因下调了NOD1信号通路在RAW264.7细胞中的抗烟曲霉孢子刺激作用.This paper aims to study the role of NOD1 and TLR4 signal transduction in RAW264.7 monocyte-macrophages in the state of normal or immunosuppression when stimulated by Aspergillus fumigatus conidia and to investigate the expression of NOD1 signal transduction after silencing the gene of TLR4. First, we established an immune suppression model, and then assayed expression levels of NOD1, TLR4, RIP2, MyD88 mRNA and TNF-ct protein using methods of RT-PCR and Western blot, when macrophages were stimulated for 12 h by Aspergillusfumigatus conidia after tranfected for 24 h with TLR4-siRNA by using technology of RNAi. RAW264.7 cells were cultured for 12 h with RPMI1640 culture medium containing immunosuppressive agents （methylprednisolone sodium succinate） at a concentration of 0.04mg/mL. The immune suppression model of macrophages was established with 90% survival rate. The signaling pathway of NOD1 and TLR4 in macrophages was activated by given stimulus of Aspergillus fumigatus conidia and exerted the effect of anfi-Aspergillus fumigatus spores stimulation through releasing pro-inflammatory cytokines TNF-α. The macrophages cannot start their effective immune response when they were in the state of immunosuppression. Silencing efficiency of macrophates was up to 83% after transfection with TLR4-siRNA. Silencing the gene of TLR4 down-regulates the effect of NOD1 signal transduction in RAW264.7 cells on anfi-Aspergillusfumigatus conidia stimulation.

关 键 词：NOD1 TLR4 TLR4-siRNA 烟曲霉孢子 调控关系 

分 类 号：R392.1[医药卫生—免疫学]