【作者】 王晓东；

【导师】 叶放蕾；

【作者基本信息】 郑州大学 ， 耳鼻咽喉科学， 2013， 硕士

【摘要】 背景耳聋是指听觉系统中传音部分或(和)感音部分及其听觉传导通路中听神经及各级听觉中枢发生病变所引起的不同程度的听力下降,是耳科疾病三大症状之一。据世界卫生组织1996年估计,听力损失者近6亿。根据我国2006年进行的全国第2次残疾人抽样调查结果显示,听力残疾人约2004万人,言语残疾127万人,共占各类残疾人总数的27%,严重影响了人们的生活和日常工作。因此,耳聋的预防和治疗仍是耳科医师和耳科学工作者肩负的神圣使命。随着分子生物学、听力学、电生理学和耳显微外科技术的迅猛发展,对大多数传导性聋目前可通过手术治疗提高听力。但是对于感音神经性聋的治疗,仍是目前困扰耳科界的最大难题之一。感音神经性聋(sensorineural deafness, SNHL)最常见的原因是外周听觉系统中的耳蜗毛细胞丧失,引起毛细胞缺失的主要诱因是机械性听觉感受器的损伤、噪声暴露引起的内耳变化、耳毒性药物如氨基苷类抗生素引起的听觉感受器的损伤、老化期间出现的退行性变化等。这些病理变化后听觉中枢系统的改变具有某些程度的相似性,简而言之,各种原因引起的听力损失所致的听觉中枢的可塑性变化是相似的。正是由于这种可塑性的存在,为听觉剥夺的患者提供了恢复听觉功能提供了可能。现在,随着世界上老龄化人口和噪音污染日益增加,耳聋患者也逐渐增加,这迫切需要寻找一条治疗耳聋的新思路,听觉中枢的可塑性恰恰为耳科医师及耳聋患者提供了希望,进一步理解听觉中枢系统的可塑性更具有现实意义。研究发现,听觉剥夺早期可导致听觉中枢系统在形态和功能上发生相应的适应性改变。通过研究这些改变,有助于我们进一步认识听觉中枢的功能。本实验中,建立了听觉剥夺小鼠的动物模型,并应用免疫组化观察听觉剥夺后脑干听觉中枢声信号重塑相关蛋白的变化,以达到进一步了解后天性聋对听觉中枢影响的目的。目的1.通过损毁耳蜗建立后天性聋动物模型,为听力损失后听觉中枢的可塑性研究提供稳定、可靠的途径。2.通过研究小鼠单侧耳蜗损毁后耳蜗核(cochlear nucleus, CN)和下丘(inferior colliculus, IC)中生长相关蛋白.-43(growth-associated protein gap-43, GAP-43)在术后不同时间点的表达变化,探讨小鼠听觉中枢在听觉剥夺后突触的重塑和修复过程。材料和方法1.建立后天性聋动物模型。参照田永胜[1]等的“小鼠听觉剥夺动物模型的建立”一文中的方法建立成年小鼠听觉剥夺动物模型。选用2-3月龄的昆明小鼠30只,体重25-35g,健康、无外耳道和中耳感染,耳廓反应灵敏(河南省实验动物中心提供)。所用实验动物均无耳毒性药物使用及强噪声暴露史。按照随机原则将实验动物分为6组,分别为正常对照组和单侧耳蜗损毁后3、7、15、30、60天组,每组各5只。单侧耳蜗损毁组在大视野棱镜式手术放大镜下,用显微手术器械切除鼓室后壁,辨清耳蜗位置后,损毁耳蜗。2.HE染色观察正常对照组和单侧耳蜗损毁组耳蜗核和下丘神经元形态的改变。3.采用免疫组化学SP法对正常对照组和耳蜗损毁不同时间点耳蜗核和下丘GAP-43蛋白的表达进行检测,并用计算机图像分析系统定量分析。采用SPSS17.0分析软件进行统计学分析,各组间差异用单因素方差分析,两组间的比较用独立样本t检验或校正t检验,多组间的两两比较用LSD-t检验。检验水准α=0.05,P<0.05,为差异有统计学意义。结果1.建立了稳定的听觉剥夺小鼠模型。2.耳蜗切除组术侧耳蜗核和双侧下丘中部分神经元出现体积萎缩,突起消失,胞浆着色浅淡,核固缩、碎裂或消失。3.小鼠单侧耳蜗损毁后3天、7天、15天,术侧耳蜗核GAP-43含量水平呈上升趋势,较正常对照组及非术侧表达升高(P<0.05)；术后30天,GAP-43表达水平下降,但表达水平仍高于正常对照组,差异有统计学意义(P<0.05),到损毁后60天,GAP-43表达水平稍高于正常对照组,差异无统计学意义(P>0.05)。非术侧耳蜗核GAP-43含量水平与正常对照组相比,差异无统计学意义(P>0.05)。4.小鼠单侧耳蜗损毁后3天,术耳同侧和对侧下丘GAP-43表达水平较正常对照组升高,差异具有统计学意义(P<0.05),术耳同侧和对侧差异也具有统计学意义(P<0.05)；术后7天、15天,表达均持续上升；术后30天、60天表达逐渐下降,但术耳同侧和对侧仍高于正常对照组(P<0.05),除正常组及60天组外,双耳差异有统计学意义(P<0.05)。结论1.切除耳蜗可建立稳定可靠的听觉剥夺动物模型。2.单侧耳蜗损毁去传入损伤后,耳蜗核和下丘核GAP-43的表达呈现出显著的动态变化过程,可能反映了脑干听觉中枢神经元在听力损失后轴突再生及突触重塑情况。更多还原

【Abstract】 BackgroundDeafness is the different levels of hearing loss caused by the lesions of conductive part or (and) sensory part in hearing system,and hearing nerve and each hearing system centre in the auditory pathway,and is one of the three major symptoms in otology diseases.According to the estimation of the world health organization in1996,there are near600million people with hearing lossing.According to the second national sampling survey for the disabled in2006,the number of hearing disabled and hearing speech disabled were respectively20.04million and1.27million,which accounted for27%of the total number of all kinds of people with disabilities.It seriously affected people’s daily life and work.Therefore, the prevention and treatment of deafness is still the sacred mission to the otology doctor and otology workers. With the rapid development of molecular biology, audiology, electrophysiology and ear microsurgery technology, most of the conductive deafness had been improved there hearing through surgery.However, the treatment of sensorineural deafness,is still one of the biggest problems in otology at present.The loss of cochlear hair cells in the peripheral auditory system is the most common cause of sensorineural deafness (sensorineural deafness,SNHL).The principal cause of cochlear hair cells loss is caused by the mechanical damage to the auditory receptors, or the changes of inner ear caused by noise exposure,the damages to the auditory receptors caused by ototoxic drugs such as aminoglycoside antibiotics,degenerative changes during the period of aging,etc. The changes of the auditory system have a certain degree of similarity after these pathological changes.In short,the plasticity changes of auditory center caused by hearing loss of all reasons are similar. Because of the existence of this plasticity, auditory deprivation patients may restore their auditory function. Now with the growing aging population and noise pollution in the world, deafness patients are also gradually increasing, and it is an urgent need to find a new idea of treatment of deafness.The auditory plasticity justly offers hope for otology doctor and deafness patients.To further understanding the plasticity of the central auditory system has more realistic significance.Studies have found that early auditory deprivation led to adaptive change in morphology and function of the auditory system. Through studying these changes, it may help us to further know the auditory function. In this experiment, the auditory deprived of mice model was established, and immunohistochemical was applied to observe the changes of acoustic signal reshape related protein in brain stem auditory center after auditory deprivation, in order to achieve further understanding of hearing center changes after acquired deafness.Purpose1. Through the establishment of animal model of acquired deafness by cochlea damaged,it provided a stable and reliable way to the study of the plasticity of the auditory center after hearing loss.2.To study the changes of growth-associated protein gap-43in cochlear nucleus and the inferior colliculus (inferior colliculus, IC) in mice after unilateral cochlear damage in different time points after surgery, and explore the remodeling and repair process of synapse in brain stem auditory center of mice after auditory deprivation.Materials and methods1.To establish animal model of acquired deafness.Prepare unilateral cochlear animal model in mice refering to Tian Yongsheng[1] about establishing "auditory deprived animal model in mice".To choose30Kunming mice(provided by experimental animal center of Henan province) with2-3months of age, weight25-35g, health, free of external auditory canal and middle ear infection, responsive auricle.All the experimental animals were used without using of ototoxic drugs and strong history of noise exposure. According to the principle of random, animals were divided into6groups:control group and after cochlear damage3,7,15,30,60days group respectively,each group of5only. Under surgical magnifier prism group in large type, remove the tympanic cavity with microsurgery instrument to figured out the cochlear position, and damage the cochlea.2. HE staining to observe the changes of nucleus cochlear and hypothalamic neurons form in the unilateral cochlear damage group and the normal control group.3. Using immunohistochemical technique SP method to detect the expression of GAP-43in cochlear nucleus and inferior colliculus in the control group and different time points after cochlea damaged,and using computer image analysis system to make quantitative analysis.Using SPSS17.0software for statistical analysis, the difference between each group used analysis of variance, the comparison between the two groups with independent samples t-test or calibration pairwise t-test,comparisons between groups with LSD-t test. Inspection level of a=0.05, P value of less than0.05was considered significant.Results1. Stable auditory deprivation mice model was established.2.Part of neurons of operation side cochlear nucleus and bilateral inferior colliculus after cochlear damaged appear volume contraction, protuberant disappear, pale cytoplasm staining, nuclear pyknosis and cataclastic or disappear.3.After the unilateral cochlear ear damaged3,7,15days, GAP-43of operation side cochlear nucleus were expressed on the up trend, and the the expression level significantly increased compared with normal control group and non-operated side.The difference was statistically significant(P<0.05).After30days postoperatively, GAP-43expression level decreased,but the expression level was still higher than normal control group, and the difference was statistically significant (P<0.05). After60days postoperatively, GAP-43expression level was slightly higher than that of normal control group,compared with the normal control group in the level of GAP-43expression,there was no statistically significant difference (P>0.05). The level of GAP-43were expressed no obvious change after surgery in the contralateral cochlear nucleus,and compared with normal control group, there was no statistically significant difference(P>0.05).4. After3days postoperatively, GAP-43expression levels of operated side and and non-operated side of the inferior colliculus were higher than normal control group, and the difference was statistically significant (P>0.05), and the difference between the operation side and the non-operated side was statistically significant(P>0.05); After7,15days postoperatively, expression levels were rising; After30,60days postoperatively, expression gradually decreases,but the operation side and the non-operated side was still higher than normal control group(P<0.05),except normal control group and60days postoperatively,the difference between the operation side and the non-operated side was statistically significant (P<0.05).Conclusions1. Removing the cochlea can establish stable auditory deprived animal model.2.After damaging the incoming hurt of unilateral cochlear,GAP-43were expressed an obvious dynamic change process in cochlear nuclei and nucleus colliculi inferioris.It may reflect the axonal regeneration and synaptic plasticity after hearing loss in brainstem auditory neurons.更多还原