TLR4基因敲除小鼠对噪声性耳蜗损伤的反应  

Effects of TLR4 Gene Knockout on Noise-Induced Cochlear Injury in Mice

作　　者：杨卫平[1,3] 许阳 胡博华[3] 杨仕明 YANG Weiping;XU Yang;HU Bohua;YANG Shiming(Department of Otolaryngology and Head&Neck Surgery,Institute of Otolaryngology,Chinese PLA General Hospital,Beijing,100853 China;Department of Respiratory Diseases,Chinese PLA General Hospital,Beijing,100853 China;Center for Hearing and Deafness,State University of New York at Buffalo,Buffalo,NY 14214,USA)

机构地区：[1]解放军总医院耳鼻咽喉头颈外科,耳鼻咽喉研究所,北京100853 [2]解放军总医院呼吸科,北京100853 [3]美国纽约州立布法罗大学听力及聋病研究中心,NY 14214 USA

出　　处：《中华耳科学杂志》2019年第6期947-952,共6页Chinese Journal of Otology

基　　金：国家自然基金资助项目(No:30973304);美国NIH资助项目(NIDCD RO1DC010154)~~

摘　　要：目的揭示Toll样受体(Toll-like receptor,TLR)基因缺失对噪声性耳蜗感觉细胞损伤,听觉功能障碍和耳蜗免疫活力的影响作用,探讨噪声性耳蜗损伤的免疫炎性机制。方法将TLR4基因敲除(TLR4 KO)小鼠接触持续噪声暴露(1-7kHz,120dB SPL)1小时,以噪声暴露的野生型(WT)小鼠为对照,检测噪声暴露前和噪声暴露后25天四个频率短纯音(4 kHz、8 kHz、16 kHz和32 kHz)诱发的小鼠双耳ABR阈值。噪声暴露后1天,25天处死动物,解剖取双侧耳蜗。采用白细胞共同抗原(CD45)荧光免疫抗体标记噪声暴露前和噪声暴露后1天耳蜗基底膜免疫细胞,鬼笔环肽(phalloidin)染色噪声暴露前和噪声暴露后25天耳蜗基底膜毛细胞纤毛、表皮板的丝状肌动蛋白。荧光显微镜下观察噪声暴露后TLR4 KO小鼠和WT小鼠耳蜗基底膜组织的巨噬细胞、单核细胞和毛细胞形态变化,计数耳蜗基底膜外毛细胞的缺失数目。结果噪声暴露后1天,WT和TLR4 KO小鼠耳蜗基底膜均呈现单核细胞渗入。噪声暴露后25天,不同频率短纯音诱发的TLR4 KO小鼠ABR阈移和耳蜗基底膜外毛细胞缺失数目均低于WT小鼠(F=71.590, df=1,90, P<0.001;Tukey test, P<0.001),(F=8.996, df=1,17, P=0.008;Tukey test, P=0.008)。结论噪声暴露后TLR4基因缺失没有阻止单核细胞渗入耳蜗基底膜,但减轻噪声暴露后耳蜗感觉细胞和听功能损伤的程度。Objective To investigate the effects of toll-like receptor(TLR) gene deficiency on noise-induced cochlear sensory cell injury, auditory dysfunction and cochlear immune activity, especially the immune inflammatory mechanism of noise-induced cochlear injury. Methods C57 BL/6 J(wild-type, WT) mice and toll like receptor 4 knockout(TLR4 KO) mice were exposed to a broadband(1-7 kHz) noise at 120 dB SPL for 1 hour. Auditory brainstem responses(ABR) to tone bursts at 4, 8, 16 and 32 kHz were measured, and missing outer hair cells were quantified before and 25 days after noise exposure. Leukocytes were visualized using CD45 immunolabeling(a receptor tyrosine phosphatase presenting on all hematopoietic/bone marrow derived leukocytes beneath the basilar membrane of the cochlea) before and 1 day after noise exposure. Results CD45 staining revealed that tissue macrophages were present beneath the basilar membrane under physiological conditions. Monocytes infiltrated into the region of the basilar membrane 1 day after noise exposure in both WT and TLR4 KO mice. At 25 days post-noise exposure, TLR4 KO mice exhibited a significant improvement of ABR thresholds at all tested frequencies(P<0.001), and the average number of missing outer hair cells(OHCs) in noise-injured ears was significantly less than in WT mice(P=0.008). Conclusion TLR4 knockout does not prevent the infiltration of monocytes into the cochlea, but reduces the level of sensory cells damage and cochlear dysfunction following noise exposure.

关 键 词：TLR4基因敲除小鼠 噪声 耳蜗 毛细胞 单核细胞 

分 类 号：R764[医药卫生—耳鼻咽喉科]