自身免疫性内耳病的实验研究

用同种异体内耳抗原免疫豚鼠，观察听阈、血清免疫学、形态学及免疫病理学的改变，结果发现分豚鼠ＡＰ（Ｎ１）反应阈明显升高，血清中出现抗内耳自身抗体，且表现有膜迷路积水，蜗轴血管炎、螺旋神经元细胞空泡变性、细胞数减少，特别是部分动物蜗轴血管和血管纹毛细血管内皮有ＩｇＧ沉积。提示通过这种实验方法可在部分动物成功地建立实验性自身免疫性内耳病动物模型。此外，就该模型与该病临床表现的相似性进行了比较。     

自身免疫性内耳病的实验研究

用同种异体内耳抗原免疫豚鼠，观察听阈、血清免疫学、形态学及免疫病理学的改变，结果发现部分豚鼠ＡＰ（Ｎ＿１）反应阈明显升高、血清中出现抗内耳自身抗体，且表现有膜迷路积水、蜗轴血管炎、螺旋神经元细胞空泡变性、细胞数减少，特别是部分动物蜗轴血管和血管纹毛细血管内皮有ＩｇＧ沉积。提示通过这种实验方法可在部分动物成功地建立实验性自身免疫性内耳病动物模型。此外，就该模型与该病临床表现的相似性进行了比较。     

内淋巴囊同种抗原免疫致自身免疫性Meniere病的实验研究

采用同种粗制内耳抗原（ＣＩＥＡｇ）在已致敏的豚鼠内淋巴囊（ＥＳ）局部免疫，诱发出明显的前庭和听觉功能障碍，以及与Ｍｅｎｉｅｒｅ病极为相似的内耳膜迷路水肿改变。从而证明ＥＳ局部同种内耳抗原免疫是一种有效的造成Ｍｅｎｉｅｒｅ病的方法，提示部分Ｍｅｎｉｅｒｅ病的发病机制可能与自身免疫因素有关。     

酶联免疫吸附试验检测抗耳蜗及蜗神经组织相关抗体的应用

自制牛耳蜗抗原（ＣＣＡｇ）及牛蜗神经抗原（ＣＣＮＡｇ）采用酶联免疫吸附试验（ＥＬＩＳＡ）对５２例（８９侧）不明原因感音神经性聋患者及对照组２０例无耳疾和免疫性疾病的患者进行了检测，结果抗ＣＣＡｇ抗体水平对照组为０．３８１１±０．０７４３，（吸光度Ａ值，ｘ±ｓ，下同），实验组为０．９４２１±０．２４０８，两组差异有高度显著性（Ｐ＜０．０１），以对照组抗ＣＣＡｇ抗体Ａ值的ｘ＋２ｓ判断，实验组２４例抗ＣＣＡＧ抗体阳性，阳性率为４６％。抗ＣＣＮＡｇ抗体水平对照组为０．２２５４±０．０３８２，实验组为０．２３３１±０．０６５６，两组差异无显著性，以ｘ＋２ｓ判断，实验组６例抗ＣＣＮＡｇ抗体阳性，阳性率为１１．５％。认为ＥＬＩＳＡ检测法程序简便，可动态观察患者抗体水平，并能定量分析，配合免疫组织化学方法或免疫转印法可用于内耳自身免疫病的诊断。用ＥＬＩＳＡ法检测到抗ＣＣＮＡｇ抗体，证实了国内外学者的推断，即内耳自身免疫病存在蜗后起源的可能性。     

中线恶性网织细胞增生症的病理及免疫表型研究

目的；了解恶性网织细胞增生症（ＭＭＨ）的病理形态学改变，从免疫表型的研究角度进一步阐明ＭＭＨ中异型淋巴细胞（ＡＬＣ０的来源属性，探讨ＭＭＨ与恶性淋巴瘤的关系。方法：对确诊的３９例ＭＭＨ病理组织切片常规ＨＥ染色，光镜下观察病理形态改变；同时行免疫组织化学染色（ＳＰ法）。结果：（１）ＭＭＨ的病理为凝固性坏死伴混合炎性细胞浸润，具有亲上皮性浸润，围绕血管中心分布和破坏血管等特征。（２）３９例ＭＭＨ中２８     

自身免疫性感音神经性聋的内耳代谢与电生理研究

为探讨自身免疫性感音神经性聋（ＡＳＨＬ）的内耳病理生理学机制，采用听觉电生理技术和酶组织化学方法，观察ＡＳＨＬ模型动物的内耳生理功能与组织内主要酶活性的变化。结果示：听视经复合动作电位和耳蜗微音器电位阈值明显升高，内淋巴电位（包括负相）幅值均有不同程度的降低，并与血管纹和内淋巴囊局部组织内Ｎａ＾＋－Ｋ＾＋－ＡＴＰ酶和琥珀酸脱氢酶活性改变之间的相关性。表明自身免疫性内耳损伤，进而造成组织内相关酶代谢     

含抗内耳抗原特异性抗体的体液转移免疫致豚鼠先天性感音神经性聋的实验研究

目的：证实针对内耳组织抗原特异性抗体通过胎盘可造成子鼠先天性自身免疫性感音神经性聋。方法：同种粗制内耳抗原（CIEAgs）免疫豚鼠,造成自身免疫性感音神经性聋（ASNHL）动物模型。ELISA法测定示其血清抗体水平升高,耳蜗电图（ECochG）示其听神经复合动作电位和（或）耳蜗微音器电位阈值或伪阈升高。取其血清（含特异性抗内耳组织抗原抗体）持续转移免疫妊娠豚鼠,采用ECochG测试被动免疫妊娠母鼠和子鼠的听觉功能,再采用颞骨火棉胶切片和苏木精一伊红染色,光镜观察内耳组织形态学改变。结果：各组出现听觉损伤的动物是：采用ASNHL模型动物血清被动免疫的8只妊娠母鼠中有5只（7耳）和其所产子鼠8只中有6只（10耳）,采用非ASNHL模型动物血清免疫的妊娠豚鼠所产子鼠5只中有1只（2耳）。出现听力损失的被动免疫母鼠和子鼠内耳病理形态学改变主要为螺旋神经节细胞变性和Rosenthal管中炎性细胞（以单个核细胞为主）浸润,部分动物出现膜迷路积水。结论：同种体液转移免疫可造成自身免疫性内耳病变,子鼠产生听觉功能障碍主要是由于母鼠所产生的特异性抗内耳组织抗原抗体经胎盘到达子鼠体内所致。     

纯化内耳P0蛋白诱发实验性自身免疫性内耳病动物模型

目的：用从内耳组织中纯化的P0蛋白免疫豚鼠,建立P0蛋白诱发的自身免疫性内耳病动物模型,研究其在自身免疫性内耳病中的作用。方法：采用制备性SDS-PAGE从内耳组织中分离、纯化P0蛋白。以纯化的豚鼠内耳P0蛋白作为抗原免疫豚鼠,观察其听性脑干反应阈,血清中抗体水平和内耳形态学的改变,并用免疫组织化学法确定P0蛋白在耳蜗的分布情况。结果：SDS-PAGE结果显示纯化的蛋白质只在分子量为30 000的位置上出现单一的蛋白染色带,Western blot结果示该蛋白即为P0蛋白。免疫后有22%豚鼠的听性脑干反应阈升高,对照组动物无变化。实验组血清IgG显著升高（F=6.48,P〈0.01）,反应阈提高豚鼠的螺旋神经节细胞均有不同程度的数目减少,蜗轴小血管周围有炎性细胞浸润。P0蛋白在耳蜗仅分布于螺旋神经节、蜗轴神经纤维的髓鞘上。结论：用制备性SDS-PAGE能够成功地从内耳纯化P0蛋白,用于自身免疫性内耳病的研究。P0蛋白在部分豚鼠能够诱发自身免疫性内耳病,可能是自身免疫性内耳病的自身抗原之一。     

喉癌相关抗原免疫电镜的定位研究

目的 研究喉癌混合单克隆抗体相关抗原的定位，为喉癌的早期诊断及导向药物治疗奠定理论基础。方法：采用三株喉癌单克隆抗体ＬＣ９、ＬＣ１１、ＬＣ１２混合对９０例病理证实的喉癌组织切片（病理Ⅰ级４９例，Ⅱ级４１）、１４例喉癌前病变及１０例正常喉粘膜进行免疫组化染色（ＡＢＣ法）光镜观察，同时采用ＬＳＡＢ法对９例新鲜喉癌组织细胞行免疫过氧化物酶标记电镜包埋前染色，对混合单克隆抗体相应抗原进行超微结构的定位观察     

一氧化氮在豚鼠耳蜗作用的实验研究

为探讨ＮＯ在内耳的作用，采用外淋巴给药途径，观察一氧化氮气体（ＮＯ）、Ｌ－精氨酸、硝普钠及一氧化氮合酶（ＮＯＳ）拮抗剂Ｎ－甲基－Ｌ－精氨酸对耳蜗蜗内电位（ＥＰ）、复合动作电位（ＣＡＰ）及耳蜗微音器电位（ＣＭ）的影响。结果表明，Ｎ－甲基－Ｌ－精氨酸可以使ＥＰ减小５０％，ＣＡＰ振幅降低３３％及ＣＭ振幅略有降低，在此基础上，用Ｌ－精氨酸外淋巴灌流可以逆转Ｎ－甲基－Ｌ－精氨酸所致的改变。ＮＯ持续外淋巴缓释能使Ｎ－甲基－Ｌ－精氨酸导致的ＥＰ、ＣＡＰ及ＣＭ的改变恢复，并超过正常，随之出现快速下降。外淋巴灌流硝普钠后，ＥＰ、ＣＡＰ及ＣＭ短暂升高后逐渐下降，并维持在较低水平。ＣＡＰＮ１波及ＣＭ潜伏期的变化规律与其振幅的变化规律基本一致。结果提示，ＮＯ在生理条件下维持内耳功能，可能参与耳蜗毛细胞微机械特性及敏感性的调节，过量表达可以产生耳蜗毒性     

三种内耳抗原与自身免疫性内耳病的相关性研究

目的 探讨3种纯化的内耳抗原与自身免疫性内耳病的关系,并确定其在耳蜗的表达。方法 以粗制内耳抗原的3种亚组分（31000、42000－45000和60000蛋白）作为抗原,分别免疫动物（B、C和D组）,观察听阈、血清IgG水平和内耳形态学的改变,并应用免疫组织化学方法确定其在耳蜗的表达。A组为对照组,以不含抗原的聚丙烯酰凝胶匀浆液代替内耳抗原。结果 免疫前各组听阈差异无显著性（F＝0．07,P＞0．05）,免疫后对照组和C组听阈和内耳形态学未见明显改变,B组（9／30只）和D组（7／28只）部分动物出现听力损失。组间听阈差异有显著性（F＝9．12,P＜0．01）。实验组动物血清IgG均显著性升高（F＝7．46,P＜0．01）,B、C和D组与对照组相比差异有显著性。31000蛋白完全分布于耳蜗神经纤维,而42000－45000和60000蛋白分布广泛,螺旋神经节、Corti器、血管纹和螺旋韧带均有分布。结论 粗制内耳抗原中31000和60000亚组份均能独立诱发自身免疫性内耳病,31000蛋白具有很高的组织特异性,可能作为一种标志性蛋白用于自身免疫性内耳病的临床诊断。     

同种内耳抗原免疫致聋豚鼠的子代内耳功能和形态学观察

目的观察自身免疫性感音神经性聋(autoimmune sensorineural hearing loss，ASHL)母鼠所产子代内耳听觉和前庭生理功能、病理形态学的变化，初步探讨母鼠体内针对内耳组织抗原免疫反应是否可以造成子代的内耳损伤。方法同种内耳抗原(homogeneous inner ear antigens，HIEAg)持续免疫孕豚鼠，采用耳蜗电图(包括听神经复合动作电位)、总和直流电位、耳蜗微音器电位和眼震电图仪(记录自发性和冷热空气试验)测试母鼠和子鼠的听觉和前庭功能，并检测针对HIEAg的血清特异性体液和细胞免疫反应，采用火棉胶切片和HE染色，光镜观察内耳病理组织学改变。结果ASHL母鼠所产子鼠中，部分(3／7)出现听觉损伤，并发现其血清中特异性抗体水平升高，内耳出现免疫炎性病理损伤。非ASHL母鼠和对照组母鼠所产子代未见明显异常。结论ASHL雌鼠所产子代可出现感音神经性聋，其内耳损伤和功能障碍极可能与针对内耳组织的自身免疫反应(尤其是体液免疫)有关。     

Relationship between three inner ear antigens with different molecular weights and autoimmune inner ear disease

Crude inner ear antigen (CIEAg) can induce autoimmune inner ear disease (AIED) although it is not known which subcomponent of CIEAg is involved. In this study, we investigated the relationship between 3 purified inner ear antigens (31, 42-45 and 60 kD proteins) and AIED, and determined their distribution in normal guinea pig cochlea. Three groups of guinea pigs were immunized with the three inner ear antigens and one group served as a control. The hearing thresholds, serum IgG level and morphological changes in the inner ear were observed. The expression of the three antigens in the cochlea was detected using immunohistochemical techniques. No obvious changes in hearing thresholds or inner ear morphology were observed between the control and 42-45 kD groups. Animals immunized with the 31 or 60 kD proteins showed a significant increase in hearing thresholds (p < 0.05 vs control), accompanied by morphological changes in the inner ear. The serum IgG level was increased significantly (p < 0.05) in all immunized animals. The 31 kD protein was distributed in the cochlear nerve and spiral ganglion, while the 42-45 and 60 kD proteins were distributed widely, being found in the spiral ganglion, organ of Corti, stria vascularis and spiral ligament. These results suggest that two subcomponents of CIEAg (the 31 and 60 kD proteins) may induce AIED independently, that several inner ear antigens may contribute to the pathogenesis of AIED and that the 31 kD protein is of high tissue specificity and may be used as a marker protein for the clinical diagnosis of AIED.     

周围神经脱髓鞘豚鼠模型听神经病变及听功能研究

目的:通过建立实验性变态反应性神经炎这一周围神经脱髓鞘动物模型,观察其听神经病变,初步探讨其听性脑干反应(ABR)和听神经复合动作电位(CAP)的改变。方法:以粗提的牛外周神经髓鞘碱性蛋白(MBP)作为抗原,免疫实验组豚鼠;对照组以生理盐水代替MBP。检测动物血清抗MBPIgG水平、坐骨神经传导速度,观察坐骨神经、听神经病理改变;检测ABR、CAP阈值及潜伏期;观察内耳病理损伤。结果:实验组血清抗MBPIgG水平升高,与对照组相比P<0.01;实验组坐骨神经传导速度减慢,与对照组相比P<0.05;透射电镜发现坐骨神经、听神经脱髓鞘改变;免疫前后实验组14只(26耳)出现ABR反应阈升高,伴Ⅰ、Ⅲ、Ⅴ波潜伏期明显延长,与对照组比较P<0.01,而Ⅰ～Ⅲ、Ⅲ～Ⅴ波间期与对照组比较P>0.05;CAPN1、N1波潜伏期延长,与对照组比较P<0.01;另有4只(8耳)仅出现潜伏期延长而无阈值升高;免疫组织化学显示内耳免疫损伤部位主要在蜗神经、内耳神经纤维、螺旋神经节;扫描电镜显示内毛细胞纤毛紊乱、胞质溢出。结论:实验性变态反应性神经炎动物模型作为一种可靠的周围神经脱髓鞘动物模型,其病变可累及听神经出现听神经脱髓鞘改变,ABR和CAP阈值升高、潜伏期明显延长,该模型可望成为探讨听神经脱髓鞘的听力学表现的一种有用的动物模型。     

先天性自身免疫性感音神经性聋防治的实验研究

目的 探讨免疫抑制剂环磷酰胺是否可以有效地预防和治疗先天性自身免疫性感音神经性聋.方法 同种粗制内耳抗原免疫雌性豚鼠,在妊娠期间给予免疫抑制剂环磷酰胺预防,对于未给予免疫抑制的母鼠所产子鼠给予环磷酰胺治疗,观察实验动物内耳听觉功能和病理形态学变化.结果 同时给予免疫抑制的母鼠及其各自所产子鼠均未出现明显的听觉损伤和内耳病理改变;出现听觉功能障碍和内耳病理变化的子鼠经环磷酰胺治疗后,其听觉功能有所提高(主要为低频区).结论 环磷酰胺可有效地预防母体内针对内耳组织特异性自身免疫反应所造成的子鼠先天性感音神经性聋,但疗效有限.     

Transsynaptic delivery of nanoparticles to the central auditory nervous system

CONCLUSION: Silica nanoparticles may serve as a nonviral delivery system to the sensory hair cells, spiral ganglion cells within the cochlea, and the vestibular organ, as well as the cochlear nucleus. OBJECTIVES: At present there are no targeted therapeutics for inner ear disease. A variety of viral vector systems have been tested in the inner ear with variable efficacy but they are still not regarded as safe systems for inner ear delivery. Nanoparticles are a nonviral method of delivering a variety of macromolecules that potentially can be used for delivery within the auditory system. In this study, we evaluated the distribution and safety of nanoparticles in the inner ear. MATERIALS AND METHODS: Cy3-labeled silica nanoparticles were placed on the round window membrane of adult mice. Hearing thresholds were determined after nanoparticle delivery by auditory brainstem responses (ABRs). Distribution of particles was determined by histological evaluation of the cochlea, vestibular organs, and brain stem. RESULTS: Fluorescent microscopy demonstrated Cy3-labeled nanoparticles signals in the sensory hair cells and the spiral ganglion neurons of both the treated and contralateral inner ears. Additionally, the distal part of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) was found to be labeled with the Cy3-linked silica nanoparticles, indicating a retrograde axonal transport. No hearing loss or inflammation was noted in the treated cochlea.     

Age-dependent degeneration of the stria vascularis in human cochleae

OBJECTIVE: Aging is a common cause of acquired hearing impairments. This study investigated age-related morphologic changes in human cochleae, with a particular focus on degeneration of the stria vascularis (SV) and the spiral ganglion (SG). STUDY DESIGN: Retrospective case review. METHODS: The study group comprised 91 temporal bones from individuals aged 10 to 85 years who had no history or audiometric findings suggestive of specific causes of cochlear degeneration. We quantified the SV and SG atrophy at each cochlear turn using morphometric measurements. Correlations of the SV and SG atrophy with age, audiometric patterns of hearing loss, and auditory thresholds were statistically investigated. RESULT: The SV and the SG both showed a tendency for progressive atrophy to develop with age. However, statistically significant correlations were observed between aging and SV atrophy only in the apical and basal cochlear turns. These findings were consistent with those reported previously in gerbils. No significant correlations were detected between SV or SG atrophy and audiometric findings. CONCLUSION: SV atrophy appears to be the most prominent anatomic characteristic of aged human cochleae.     

内耳结构异常的语前聋人工耳蜗植入儿童听觉语言康复效果评价

目的评价不同类型内耳结构异常的语前聋儿童人工耳蜗植入术后听觉语言康复效果。方法选取人工耳蜗植入儿童10例(12耳),其中内耳结构发育正常的3例,前庭导水管扩大3例,Mondini畸形1例,蜗孔狭窄1例,内听道狭窄听神经纤细2例,分别采用电刺激诱发听神经复合动作电位(electrically evoked compound action potential,eCAP)、助听听阈、听障儿童听觉能力、语言能力评估标准及方法,从听神经客观电生理水平、听察觉阈、听觉辨识与理解能力以及语言能力4方面评价患儿的康复效果。结果①客观电生理:单纯前庭导水管扩大患者听神经对电刺激敏感度与内耳发育正常者相似,Mondini畸形患者比内耳发育正常者略高,蜗孔狭窄及听神经纤细患者听神经敏感度较差;②听察觉阈:与内耳结构正常及轻度畸形(Mondini畸形)患者相比,蜗孔狭窄及听神经纤细患者听察觉阈偏高,高频比低频阈值低;③听觉辨识与理解能力:在安静环境近距离交谈情境下,单纯前庭导水管扩大、Mondini畸形及蜗孔狭窄患者对熟悉语词和短句的辨识与理解能力与内耳发育正常者无差别,听神经纤细患者得分较低;④语言能力:单纯前庭导水管扩大、Mondini畸形患者与内耳发育正常者无差别,蜗孔狭窄患者的语言交往与表达能力落后于内耳发育正常者,听神经纤细患者在语法、理解、交往、表达4个维度均落后于内耳发育正常者。结论语前聋儿童人工耳蜗植入术后听觉语言康复效果需要使用多种方法综合评价。Mondini畸形患者给予足够电刺激量后,其听觉语言能力可达到内耳发育正常者水平。蜗孔狭窄、听神经纤细等严重内耳畸形患者,人工耳蜗对其听觉语言能力发展有一定帮助,家长需要建立合理期望值并坚持长期康复。     

The protective effects of ciliary neurotrophic factor on inner ear damage induced by intensive impulse noise]

OBJECTIVE: To evaluate the feasibility of using ciliary neurotrophic factor(CNTF) to treat intensive impulse noise-induced inner ear damage. METHODS: The guinea pigs were given either CNTF (CNTF group) or 0.9% sodium chloride (NS group) for 3 weeks after impulse noise exposure. The animals receiving neither medicine nor noise served as a control group. ABR threshold shifts, the cochlear AchE staining as well as the hair cell and spiral ganglion cell counting were carried out in three groups of animals. RESULTS: The numbers of damaged hair cells and spiral ganglion cells in the CNTF group was less than that in the NS group. AchE activity alteration was also less severe in the CNTF group. Similar to the morphological results, changes in the auditory function, represented by the ABR threshold shifts, was less in the CNTF group. CONCLUSION: CNTF can protect cochlear hair cells and spiral ganglion cells against intensive impulse noise exposure by decreasing degeneration and necrosis of the hair cells in some extent and expedite hearing recovery.