Toll样受体2和4在小鼠肺炎链球菌中耳急性感染中的作用  

Toll-like receptor 2 and Toll-like receptor 4 participates in mediation of acute otitis media and mortality in pneumococcal infections in mice

作　　者：李胜利[1,2] 张敏燕 李白芽 郑庆印 朱宏亮[4] 

机构地区：[1]西安交通大学医学院第二附属医院科研实验中心,710004 [2]第二炮兵总医院耳鼻咽喉头颈外科 [3]美国西储地大学医学院耳鼻咽喉头颈外科 [4]西安交通大学神经科学研究中心

出　　处：《中华耳鼻咽喉头颈外科杂志》2011年第12期1009-1018,共10页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：国家自然科学基金（39970785,81170922）;国家自然科学基金国际合作项目（30210103151）

摘　　要：目的 探讨Toll样受体2（Toll-like receptor 2,TLR2）和Toll样受体4（Toll-like receptor 4,TLR4）在小鼠肺炎链球菌中耳感染中的作用.方法 野生型（wild type,WT）C57BL/6J小鼠、TLR2缺陷（TLR2-/-）和TLR4缺陷（TLR4-/-）小鼠分别通过中耳鼓膜接种肺炎链球菌悬液[1×106菌落形成单位（colony forming unit,CFU）].在细菌接种前、接种后第3天和第7天分别进行听性脑干反应（ABR）和鼓室声导抗测试,血液及中耳积液中细菌滴度测定,颞骨病理切片形态学观察,反转录聚合酶链反应（RT-PCR）检测不同基因型小鼠炎性细胞因子IκB激酶β（IκB kinase β,IKKβ）、核因子κB（NF-κB）、肿瘤坏死因子α（TNF-α）、白细胞介素-1β（interleukin-1β,IL-1β）、巨噬细胞炎性蛋白1α（MIP-1α）、黏蛋白/黏液素5AC和5B（MUC5AC和MUC5B）mRNA的表达.结果 中耳接种肺炎链球菌3d后,TLR2-/-小鼠死亡率为58.8％（40/68）,TLR4-/-小鼠死亡率为35.6％（21/59）,而WT小鼠的死亡率仅为17.3％（9/52）.接种7d后,TUR2-/-小鼠死亡率为82.4％（54/68）,TLR4-/-小鼠死亡率为54.2％（32/59）,而WT小鼠的死亡率仅为23％（12/52）,三组之间差异具有统计学意义（P＜0.01）.ABR测试结果显示,接种后存活3d及7d的TLR2-/-和TLR4-/-小鼠ABR阈值高于WT小鼠,三组之间的差异具有统计学意义（P＜0.01）.颞骨病理发现,TLR2-/-和TLR4-/-小鼠接种肺炎链球菌后第3天中耳出现积液和组织破坏,接种后第7天感染极为严重.TLR2-/-鼠出现严重的耳蜗炎性细胞浸润,内外毛细胞破坏、盖膜肿大和血管纹退变,以及螺旋神经节细胞的损伤；TLR4-/-鼠可见耳蜗内外毛细胞和螺旋神经节细胞的损伤；而WT鼠的耳蜗未见炎性细胞浸润和组织破坏,内外毛细胞基本正常.在接种细菌3d和7d的TLR2-/-鼠中耳黏膜未发现肥大细胞,但在TLR4-/-和WT鼠中耳黏膜发现较多的肥大细胞并有脱颗粒现象.接种肺炎链球菌后3d和7Objective To investigate the roles of Toll-like receptor 2（TLR2）and Toll-like receptor 4（TLR4）in host defense against Streptococcus pneumoniae infection in the middle ear.Methods Wild-type（WT）C57BL/6J,TLR2-deficien（TLR2-/-）and TLR4-deficien（TLR4-/-）mice were inoculated with Streptococcus pneumoniae（1 × 106CFU）through the tympanic membrane.All animals were tested the mouse ABR thresholds and tympanometry measurement before,and 1 day,3 days and 7 days following pneumococcal challenge.Blood bacterial titer were determined by plating 50 μl volumes of 10-fold diluted blood.Histological analysis of middle ear and inner ear were performed by fixation,decalcification,embedded section,and counterslained with hematoxylin/eosin and toluidine blue staining.Semi-quantitative RT-PCR was applied to determine mRNA accumulation of TLR2 and TLR4 related genes.Results Forty of 68 TLR2-/-mice and twenty-one of 59 TLR4-/-mice showed bactermia and died within 3 days after the pneumococcal challenge,however,only 9 of 52 WT mice died.The survive mice were shown have more severe heating loss in the TLR2-/-and TLR4-/-mice than in the WT mice,indicated by ABR thresholds,at 3 or 7 days postinoculation.The histological pathology was characterized by effusion and tissue damage in the middle ear,and in the TLR2-/-and TLR4-/-mice,the outcome of infection became more severe at 7 days.At both 3 and 7 days after challenge,the TLR2-/-mice had higher blood bacterial titers than WT mice（P 〈 0.05）.Temporal bone histopathologic change indicated that 3 days after the pneumococcal challenge,the TLR2-/-and TLR4-/-mice showed effusion and tissue damage in the middle ear,and the infection became more severe at 7 days postinoculation.TLR2-/-mice showed severe inflammatory cell infiltration in the cochlear,the organ of Corti showed the outer hair cells damage,the tectorial membrane swelling,degeneration of the stria vascularis,and severe loss of spiral ganglion cells; However,the WT mice was not found the cell infiltration and

关 键 词：TOLL样受体2 TOLL样受体4 链球菌 肺炎 中耳炎 化脓性 细胞因子类 小鼠 

分 类 号：R764[医药卫生—耳鼻咽喉科]