前庭学

991922 PCR技术对大鼠内耳膜迷路线粒体DNA的检测/杜瀚…//临床耳鼻咽喉科杂志一1999,13(/1)一176~177 目的:建立灵敏、可靠的大鼠内耳膜迷路线粒体DNA(mtDNA)提取和检测方法。方法:结合PCR技术扩增,ntl)NA编码NDI一16 Sr-RNA基因的601 bp片段,检测大鼠听泡内耳膜迷路线粒体DNA方法,并对两种mtDNA获取方法进行比较。结果:采用Seidman的方法,将1只大鼠的双侧听泡内耳膜迷路作为一个样品,检测10个样品均成功扩增出编码NDl一16 SrRNA基因的601 bp片段;而采用EdrismtDNA提取方法.需用6侧听泡内耳膜迷路作为一个样品方可获得可…     

人听觉器官线粒体DNA^4977缺失与老年聋的关系

目的 探讨ｍｔＤＮＡ＾４９７７缺失与老年聋的关系。方法 采用聚合酶链反应（ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ，ＰＣＲ）及套式ＰＣＲ技术，扩增正常及缺失区ｍｔＤＮＡ片段，ｐＧＥＭ－Ｔ Ｅａｓｙ载体连接扩增产物，重组、克隆目的ＤＮＡ进行序列分析。检测６７耳（４１例）颞骨切片、２１例蜗核（双侧）、２０例颞叶脑组织、２０例心肌和２２例肝脏组织中ｍｔＤＮＡ＾４９７７缺失的发生率，根据患者生前的临床资料分为老年组与非老年组、老年聋组与老年听力正常组进行比较分析。结果 被检所有标本均成功扩增到正常ｍｔＤＮＡ编码ｔＲＮＡ和ＮＤ１片段的ＮＡＤＨ基因。在老年人多种组织中检测到了ｍｔＤＮＡ＾４９７７缺失，老年组不同器官组织中ｍｔＤＮＡ＾４９７７缺失发生率均明显高于非老年组，两组之间的差异具有显著性意义（χ＾２检验，Ｐ     

线粒体DNA突变与感音神经性聋

线粒体ＤＮＡ(ｍｔＤＮＡ)突变与多种综合征、全身性疾病及老年退行性疾病有关[1] 。近年的研究显示感音神经性聋也是多种ｍｔＤＮＡ突变的唯一表现[2 ,3] 。本文综述ｍｔＤＮＡ突变引起的各种感音神经性聋及其研究进展。1　线粒体ＤＮＡ基因结构和特点线粒体ＤＮＡ (ｍｔＤＮＡ)是唯一存在于人体细胞质中的ＤＮＡ分子。每个ｍｔＤＮＡ分子编码、合成线粒体蛋白质必须的两类ｒＲＮＡ(12Ｓ和 16ＳＲＮＡ)、2 2种ｔＲＮＡ及 13个与细胞氧化磷酸化 (ＯＸＰＨＯＳ)有关的多肽链亚单位[1,4 ] 。ｍｔＤＮＡ为母系遗传 ,具有遗传异质性 ,ｍｔＤＮＡ…     

豚鼠内耳膜迷路蛋白电泳分析

采用十二烷基磺酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）技术分析了豚鼠内耳膜迷路的蛋白组成。结果表明：豚鼠内耳膜迷路含有６８ｋＤ以及与１３０ｋＤ、４５ｋＤ相近的蛋白条带，和文献报告的牛、人的内耳膜迷路蛋白分子量相同或相近，从而为进一步分析、纯化内耳特异性抗原，为临床采用免疫转印技术检测抗内耳组织自身抗体提供了可能。     

线粒体DNA缺失突变在氨基糖苷类抗生素耳毒易感？…

目的 建立大鼠线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌ ＤｎＡ，ｍｔＤＮＡ）缺失突变模型，探讨ｍｔＤＮＡ缺失突变在氨基糖苷类抗生素（ａｍｉｎｏｇｌｙｃｏｓｉｄｅ ａｎｔｉｂｉｏｔｉｃｓ，ＡｍＡｎ）耳毒易感性中的作用。方法 取Ｗｉｓｔａｒ大鼠随机分为Ａ、Ｂ两组，每组１５只，其中Ａ组大鼠以皮下注射阿毒素（ｄｏｘｏｒｕｂｉｃｉｎ，ＤＯＸ）２ｍｇ／ｋｇ体重，每周２次３个月，Ｂ组则以生理盐水取代ＤＯＸ，然后     

人类颞骨火棉胶切片中线粒体DNA的扩增及重组测序

目的 应用分子生物学技术建立人类颞骨火棉胶切片中的ＤＮＡ分析方法。方法 采用多聚酶链反应（ＰＣＲ）配合不同的引物、扩增方式及酶切技术检测长期保存的７例（９侧）颞骨火棉胶切片中微量线粒体ＤＮＡ的片段缺失及点突变，ｐＧＥＭ－Ｔ载体进行扩增片段的重组与克隆。结果 ９侧颞骨ＤＮＡ提取液中均可见正常的１３５ｂｐ扩增片段，巢式ＰＣＲ检出其中２例生前患老年聋者（各查１侧）有线粒体ＤＮＡ大片段缺失，测序结果证实扩     

人类颞骨火棉胶切片中线粒体DNA的扩增及重组测序

目的应用分子生物学技术建立人类颞骨火棉胶切片中的ＤＮＡ分析方法。方法采用多聚酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）配合不同的引物、扩增方式及酶切技术检测长期保存的７例（９侧）颞骨火棉胶切片中微量线粒体ＤＮＡ的片段缺失及点突变，ｐＧＥＭＴ载体进行扩增片段的重组与克隆。结果９侧颞骨ＤＮＡ提取液中均可见正常的１３５ｂｐ扩增片段，巢式ＰＣＲ检出其中２例生前患老年聋者（各查１侧）有线粒体ＤＮＡ大片段缺失，测序结果证实扩增的准确性。结论分子生物学技术在颞骨切片研究中的应用对于提高其回顾性研究水平有重要意义，目前可在耳科疾病的研究中发现相关的基因突变或病原体。     

线粒体DNA大片段缺失与老年性聋的关系

目的 :探讨内耳组织线粒体DNA(mtDNA)大片段缺失与老年性聋发病的关系。方法 :取Wistar大鼠按年龄分为A组 (青年鼠 ,4月龄 )和B组 (老年鼠 ,>2 4月龄 ) ,每组 15只。测试听性脑干反应 (ABR) ,应用聚合酶链反应 (PCR)技术对大鼠内耳膜迷路组织mtDNA 4 834bp缺失片段进行检测。结果 :A、B组大鼠ABR反应阈均值分别为 :(4 0 .0 0± 4 .6 6 )dBpeSPL、(6 4 .79± 10 .88)dBpeSPL ,二者之间差异有显著性意义 (P <0 .0 1)。大鼠内耳组织mtDNA检测发现 :A组大鼠未检测到 4 834bp缺失 ;B组大鼠有 9只存在 4 834bp缺失。 结论 :老年大鼠内耳膜迷路组织存在mtDNA片段缺失 ,这种缺失与老年性聋有关 ,可能是引起老年性聋的原因之一     

氨基糖甙类抗生素致聋家系线粒体DNA1555G点突变分析

目的考察１５５５Ｇ点突变与氨基糖甙类抗生素致聋的对应关系，建立相应的基因诊断方法提供依据。方法收集了３个有明确氨基糖甙类抗生素应用史的母系遗传耳聋家系１３人（包括聋人和听力正常者）的外周静脉血标本，聚合酶链反应扩增线粒体ＤＮＡ，Ａｌｗ２６Ｉ限制性内切酶分析、ＤＮＡ斑点杂交和ＤＮＡ序列分析检测１５５５Ｇ点突变。结果家系１和３的７份样品均为１５５５Ｇ点突变阳性，家系２的６份样品为１５５５Ｇ点突变阴性。结论发现１个１５５５Ｇ点突变阴性的氨基糖甙类抗生素致聋家系，说明线粒体ＤＮＡ１５５５Ｇ点突变不是氨基糖甙类抗生素遗传易感性唯一的分子基础。     

氨基甙类抗生素耳毒性与线粒体基因突变分析

进一步证明遗传因素对氨基甙类抗生素耳毒性的作用,探讨该病的发病机理。方法应用ＰＣＲ限制性酶切方法分析了９个氨基甙类抗生素致聋家系中６２个成员线粒体ＤＮＡ。结果发现５个家系中２０例线粒体ＤＮＡ２２ＳｒＲＮＡ基因第１５５５位Ａ→Ｇ点突变。结论该突变是氨基甙类抗生素耳毒性遗传易感性的分子生物学基础。可能有其他他粒体ＤＮＡ基因突变与氨基甙类抗生素耳毒性有关。     

迷路炎的影像学

目的 探讨迷路炎的影像学检查方法及其诊断价值.方法 观察27例31耳临床提示为迷路炎同时存在异常影像学表现的病例的高分辨率CT(high resolution CT, HRCT)及MRI图像,总结其内耳在HRCT及MRI图像上的不同改变.结果 进行了HRCT扫描的22耳中,内耳1个或多个结构表现为密度增高者6耳,密度增高且发生变形者8耳,单纯变形者1耳,内耳迷路内腔局部或完全硬化消失者7耳.在22耳中除上述改变外亦有内耳骨质缺损者4耳.进行了HRCT及MRI检查的9耳中,7耳在HRCT上可见内耳不同程度的异常改变,2耳内耳未见异常改变.MRI图像上9耳均可见内耳迷路内腔1个或多个结构T2WI信号减低或消失.行增强扫描的6耳中,4耳可见明显强化,2耳未见强化.31耳中,耳蜗受累30耳,其中仅基底周受累5耳,中顶周受累2耳,耳蜗各周均受累23耳;半规管受累26耳;前庭受累20耳;前庭窗受累18耳;蜗窗受累19耳.结论 HRCT可以较好显示骨迷路迷路炎的异常改变,MRI对于显示迷路内腔有重要价值,二者在迷路炎的影像检查与诊断中的作用是互补的,对迷路炎的临床诊断起着重要的辅助作用.     

高分辨率CT容积重建对内耳畸形的诊断价值

目的：通过高分辨率CT（HRCT）容积重建（VR）对正常内耳形态的观察,探讨其对先天性内耳畸形的诊断价值。方法：对10例（20耳）无耳部疾患者（对照组）和7例（11耳）先天性内耳畸形患者（病变组）行HRCT扫描,利用容积漫游技术对内耳骨迷路重建。对照组观察内耳骨迷路的正常结构,病变组观察内耳畸形情况。结果：正常耳VR图像不但显示了内耳骨迷路的细微结构,而且还反映了各结构之间的关系;病变耳立体显示了畸形部位及程度。11耳患耳中Mondini型7耳次;前庭及半规管畸形3耳次;前庭导水管扩大7耳次,其中6耳次伴随其他畸形;内耳道畸形2耳次且均伴随其他畸形。11耳畸形中9耳HRCT横断面图像和平面重建（MPR）冠状位图像、VR图像均可以清晰地显示畸形的部位和程度,其中VR图像可以直观、立体地显示畸形的空间形态结构;2耳水平半规管短小畸形患者VR图像较断面图像更好地显示了畸形的部位和程度。结论：VR三维重建可以立体显示正常内耳骨迷路的形态,直观显示内耳畸形的程度及病变位置,对内耳畸形的诊断具有重要的辅助价值。     

自身免疫性内耳病动物模型的抗内耳特异性抗体谱系分析

目的 为明确哪几种膜迷路蛋白成分参与了实验性自身免疫性内耳病（ａｕｔｏｍｍｕｎｅｉｎｎｅｒ－ｅａｒ ｄｉｓｅａｓｅ，ＡＩＥＤ）的形成。方法 以同种内耳组织抗原免疫豚鼠，建立自身免疫性内耳病动物模型。用免疫转印迹技术（Ｗｅｓｔ－ｅｒｎ ｂｌｏｔ）检测动物血液中抗膜迷路蛋白抗体。结果 豚鼠膜迷路蛋白成分复杂，ＳＤＳ－ＡＰＧＥ电泳至少可分出５种，但免疫后大多数动物（１５／２０）体内仅产生了抗６８ｋＤ和１     

Ototoxicity of otic drops applied to the middle ear in the chinchilla

Previous studies have shown that the application of topical otic drops to the external ear canals of animals with patent tympanostomy tubes may result in hearing impairment and cochlear hair cell loss. Otic drops are used in patients with tympanostomy tubes or tympanic membrane perforations and could have deleterious effects on the human membranous labyrinth. This report describes the inner ear damage that occurred after direct application of aminoglycoside-containing otic drops to the middle ears of experimental animals. The membranous labyrinths of 25 chinchillas were studied two days to five months after application of Cortisporin otic suspension (which contains neomycin, polymyxin B, hydrocortisone, and propylene glycol) to the middle ear cavity. Application of 0.5 ml of Cortisporin resulted in degeneration of all inner and outer hair cells throughout the cochlea, as well as severe damage to the stria vascularis. Moderate to severe degeneration of the vestibular receptor organs was also observed. The endolymphatic sacs showed dark-staining endolymph, cellular debris, and macrophages in the sac lumina, as well as increased activity of the epithelial lining.     

Quantitative analysis of mRNA in human temporal bones

CONCLUSION: Well-preserved mRNA could be extracted from frozen human inner ears. Therefore, this study demonstrates that analysis of mRNA could be performed to study the molecular mechanisms of inner ear disorders using human specimens. OBJECTIVES: Analysis of RNA as well DNA is requisite to study the molecular mechanisms of inner ear disorders. Methods of isolating RNA from experimental animals have been established, while isolation of RNA from human inner ears is much more challenging. In the present study, we demonstrate a method by which messenger RNA (mRNA) was extracted from human inner ears and quantitatively analyzed. MATERIALS AND METHODS: COCH mRNA as well as GAPDH mRNA was extracted from membranous labyrinths dissected from three formalin-fixed and three frozen human temporal bones, removed at autopsy. The length of COCH mRNA and quantity of GAPDH mRNA was compared between the two groups by quantitative RT-PCR. RESULTS: COCH mRNA could be amplified as much as 976 bp in all three frozen specimens. By contrast, it was amplified to 249 bp in two of the three formalin-fixed specimens, with no amplification observed in the remaining. The quantity of amplifiable GAPDH mRNA in the formalin specimens was only 1% of that of the frozen specimens.     

The role of mtDNA deletion in the sensitivity to aminoglycoside antibiotic induced deafness]

OBJECTIVE: To establish an animal model of mitochondrial DNA (mtDNA) deletion and investigate the possible role of mtDNA deletion in aminoglycoside antibiotic induced deafness. METHODS: Thirty wistar rats (4 months) were randomly divided into group A and B. Doxorubicin (DOX) was subcutaneously injected at doses of 2 mg/kg twice per week for 3 months in group A and then kanamycin (KM) was intraperitoneally injected 500 mg/kg per day for 10 consecutive days. The treatments of group B were identical to group A, except normal saline was substituted for DOX. The thresholds of auditory brainstem response (ABR) were measured before and after the drug administrations. The inner ear membranous labyrinthine tissue was harvested and mtDNA was amplified to identify 4,834 bp deletion by PCR technique. RESULTS: The elevation of the mean ABR thresholds in group A(67.08 +/- 8.59) dB peSPL was significantly higher than that in group B (12.71 +/- 4.42) dB peSPL after KM administration (P < 0.001). In group A, 9 of the 15 rats demonstrated 4,834 bp mtDNA deletion. However, mtDNA 4,834 bp deletion was negative in group B animals. CONCLUSION: DOX can induce mtDNA deletion in the inner ear tissue of the rat. mtDNA deletion in the inner ear may play an important role in the hypersensitivity to aminoglycoside antibiotic ototoxicity.     

The fine structure of lipofuscin in the human inner ear.

Lipofuscin inclusions in the human membranous labyrinth were studied by electron microscopy. Lipofuscin is morphologically an irregularly shaped, membrane-bound inclusion consisting of an electron-dense structure. The most common component was a fine, granular, osmiophilic substance which was always associated with a homogenous, spherical structure resembling a lipid droplet. The combination of these two components was frequently observed in the human inner ear. Distended inclusions containing lipofuscin components were also observed within the supporting cells, saccular, utricular and ampullar wall, the epithelial cells of the transitional zone and in the dark cells. Lipofuscin is closely associated with lysosome and is known to accumulate in the tissue as a result of aging. The high lysosomal activity possibly may result in lipofuscin formation in the human inner ears. Also some other unknown metabolic conditions may provide the deposits of lipofuscin.     

Identification of previously "undetectable" abnormalities of the bony labyrinth with computed tomography measurement

BACKGROUND: In patients with congenital sensorineural hearing loss (SNHL), a computed tomography (CT) scan of the temporal bone identifies inner ear malformations in approximately 25%, whereas the inner ear is grossly normal to visual inspection in the remaining 75% of the patients. In the latter group, the hearing loss is often attributed to radiologically undetectable abnormalities of the membranous labyrinth. However, subtle bony malformations may be missed because visual inspection alone is insensitive for detection. OBJECTIVE: To test the hypothesis that there are subtle bony abnormalities of the inner ear in patients with SNHL who are radiologically deemed to have normal otic bone, using standardized measurements of the inner ear. STUDY DESIGN: Retrospective review. METHODS: Measurements of the cochlea, vestibule, and semicircular canals (SCCs) were made on axial and coronal temporal bone CT scans on 15 patients with normal hearing and 15 patients with congenital SNHL and grossly normal temporal bone CT scans. Student's t-test was performed to compare the measurements of the two groups.RESULTS All studies from the SNHL group were deemed normal by visual inspection and standardized measurements (+/-2 SD from normal). Surprisingly, there were significant differences in the measurements of the cochlea and of the SCCs between patients with and without SHNL (P <.05). CONCLUSIONS: As a group, patients with SNHL and a "normal CT scan" have significant differences in the dimensions of the inner ear. This suggests that these patients have disturbed morphogenesis of both membranous and bony labyrinth. This novel observation has important implications for understanding the etiology of SNHL.     

内耳病理学研究技术的进展

内耳病理学是一门从组织形态学角度研究内耳疾病的发生原因和发展过程及其损害机制的边缘医学基础学科。内耳病理学的样品制备观察技术包括颞骨切片技术、膜迷路切片技术、耳蜗和前庭膜迷路取材铺片技术、内耳组织学和组织化学技术、内耳扫描电镜和透射电镜样品制备技术、以及电镜下的超微细胞化学技术等。通过分析各项内耳病理研究技术的发生、发展和现状，并结合实践经验和技术革新经验，深入讨论上述各项技术的优缺点和技术细节