斑马鱼内耳感觉上皮分离术

目的 探索斑马鱼内耳感觉上皮的分离方法,为采用斑马鱼作为内耳发育及疾病研究的动物模型提供技术手段.方法 取4个月龄斑马鱼进行内耳组织冰冻切片,HE染色确定内耳感觉囊斑部位,并行免疫荧光染色观察毛细胞纤毛.在解剖显微镜下将斑马鱼内耳椭圆囊、球囊及听壶完整分离,取出内耳感觉囊斑平铺于载玻片中,进行免疫荧光染色,观察内耳感觉囊斑铺片的形态结构.结果 冰冻切片证实斑马鱼内耳感觉上皮位于囊斑内,并与耳石相对应.以耳石为标志分离出完整的内耳感觉囊斑组织,铺片免疫荧光染色后可观察到内耳感觉上皮形态结构完整,毛细胞纤毛显示清晰.结论 通过内耳感觉囊斑铺片技术可以完整分离出斑马鱼的内耳感觉上皮组织.     

前庭学

970618哺乳动物前庭迷路的体外发育:周围性眩晕的研究手段/周祥宁…//天津医药一1 996,24(l)一4一7 首次在国内报告小鼠前庭迷路的体外发育。采用周祥宁和Van De Water建立的HEMA水凝胶器官培养法培养第12一13天CBA/C57小鼠胚胎的内耳原基7一8天。内耳原基的上部发育成3个半规管、椭圆囊和球囊。半规管的壶腹晴由一排感觉毛细胞和2一3排支持细胞构成。晴的上方有正在发育的峪顶。椭圆囊斑和球囊斑的表面都有一层耳石膜。这些囊斑内可见一层感觉毛细胞和1一2层支持细胞。超微结构研究发现壶腹峪、椭圆囊斑和球囊斑的毛细胞有排列规则的…     

小鼠内耳水通道蛋白的定位及其意义

目的检测水通道蛋白(waterchannelprotein;aquaporin,Aqp)亚型在小鼠内耳不同部位的分布和各亚型所在的部位。方法使用成年小鼠30只,经活体灌注,切取双侧颞骨,按石蜡包埋技术处理和切片。使用免疫组织化学方法标记确认小鼠内耳组织中水通道蛋白亚型1、3、4、5、7、9分布情况。结果Aqp-1为1∶200和Aqp-3、7、9为1∶100的一抗浓度可以看到它们在内耳不同部位稳定、清晰的染色反应,但Aqp-4和5使用1∶50甚至1∶30也未观察到染色。Aqp-1在圆窗膜、螺旋韧带、内淋巴囊和内淋巴管、椭圆囊和球囊以及内耳血管壁等处被标记;Aqp-3在螺旋韧带、前庭唇、内、外螺旋沟、基底膜和基底膜嵴、内淋巴囊和内淋巴管、膜半规管和椭圆囊、球囊斑等处显示荧光染色。Aqp-7在血管纹、基底膜、前庭膜、椭圆囊和球囊及其囊斑有反应染色,而Aqp-9在螺旋缘、前庭唇、内、外螺旋沟、前庭膜、膜半规管和球囊及其囊斑有较强染色。结论水通道蛋白亚型1、3、7、9广泛分布于小鼠内耳不同组织中,其分布部位和反应强弱存在差异,主要存在与内淋巴密切相关的组织结构中。     

Smad4 条件基因敲除小鼠前庭终器形态学研究

目的研究Smad4条件基因敲除小鼠前庭终器形忿改变，探讨Smad4基因对于前庭发育的作用。方法利用建立的Smad4条件基测敲除小鼠模型．通过光镜观察Smad4（＋／＋）、Smad4（＋／-）与Smad4（-／-）三种基因型小鼠（0．5、1．5月龄）的球囊及囊斑、椭圆囊及囊斑、半规管及壶腹嵴、前庭神经节、内淋巴管与囊的形态结构、毛细胞的形态及排列缺失情况。通过扫描电镜观察球囊斑、椭圆囊斑和壶腹嵴的形态结构、毛细胞及纤毛的排列缺失情况。结果Smad4（-／-）小鼠个体及内耳明显小于Smad4（＋／＋）和Smad4（＋／-），而后面二者区别不大。所有小鼠的球囊及囊斑、椭圆囊及囊斑、半规管、前庭神经节、内淋巴管与囊的大小和结构正常，淋巴囊腔饱满，囊斑处的感觉上皮、耳石、毛细胞及纤毛排列整齐，没有发现明显的病变．三个基因型间没有差异。Smad4（-／-）小鼠壶腹嵴囊性变多且严重、后半规管壶腹嵴出现明显的副嵴、偶尔可见壶腹嵴感觉上皮空泡样变。壶腹嵴的毛细胞和支持细胞排列整齐，形态无明显改变，未见缺失。扫描电镜示球囊斑、椭圆囊斑、后、外、上半规管壶腹嵴正常，三个基因型之间没有差异。结论Smad4（-／-）小鼠的前庭终器有轻微病理改变，Smad4（＋／＋）与Smad4（＋／-）小鼠的前庭终器形态结构基本正常．表明Smad4对于前庭终器的发育影响可能不明显。     

小鼠内耳水通道蛋白的定位及其意义

目的 检测水通道蛋白（water channel proterin;aquaporin,Aqp)亚型在小鼠内耳不同部位的分布和亚型所在部位。方法 使用成年小鼠30只,经活体灌注,切取双侧颞骨,按石虹包埋技术处理和切片。使用免疫组织化学方法标记确认小鼠内耳组织中水通道蛋白亚型1、3、4、5、7、9分布情况。结果 Aqp-1为1:200和Aqp－3、7、9为1:100的一抗浓度可以看一它们在内耳不同部位稳定、清晰的染色反应,但Aqp-4和5使用1：50甚至1：30也未观察到染色。Aqp－1在圆窗膜、螺旋韧带、内淋巴囊和内淋巴管、椭滞 和球囊以及内耳血管壁等处被标记;Aqp-3在螺旋韧带、前庭唇、内、外螺旋沟、基底膜和基底膜嵴、内淋巴囊和内淋巴管、膜半规管和椭圆囊、 球囊斑等处显示荧光染色。Aqp-7在血管纹、基底膜、前庭膜、椭圆囊和球囊及其囊斑有反应染色,而Aqp-9在螺旋缘、前庭唇、内、外螺旋沟、前庭膜、膜半规管和球囊及其囊斑有较强染色。结论 水通道蛋白亚型1、3、7、9广泛分布于小鼠内耳不同组织中,其分布部位和反应强弱存在差异,主要存在与内淋巴密切相关的组织结构中。     

前庭学

990986人前庭感觉上皮扫描电镜观察/孙建和…//中华耳鼻咽喉科杂志一1998,33(增刊)(英文版)一31一33 目的:了解人前庭器官的超微结构。方法:借助扫描电镜技术观察了5人(10耳)的前庭感觉上皮。结果:①球囊斑呈“L”形,椭圆囊斑形如打开的贝壳;②I形感觉细胞呈烧瓶样,l形感觉细胞呈试管状;③感觉细胞纤毛排列呈阶梯状,动纤毛最长,且位于最长的静纤毛一侧,椭圆囊斑最长的静纤毛和动纤毛靠近微纹,其极性也向微纹,球囊斑则背离微纹,水平半规管壶腹峙的动纤毛朝向椭圆囊侧,上、后半规管壶腹峙则朝向半规管侧;①耳石形状大小不一致,呈现两端为锥形…     

人前庭感觉上皮扫描电镜观察

为了解人前庭器官的超微结构，借助扫描电镜技术观察了５人（１０耳）的前庭感觉上皮。结果显示：①球囊斑呈“Ｌ”形，椭圆囊斑形如打开的贝壳；②Ⅰ型感觉细胞呈烧瓶样，Ⅱ型感觉细胞呈试管状；③感觉细胞纤毛排列呈阶梯状，动纤毛最长，且位于最长的静纤毛一侧，椭圆囊斑最长的静纤毛和动纤毛靠近微纹，其极性也向微纹，球囊斑则背离微纹，水平半规管壶腹嵴的动纤毛朝向椭圆囊侧，上、后半规管壶腹嵴则朝向半规管侧；④耳石形状大小不一致，呈现两端为锥形的圆柱状晶体。     

人前庭感觉上皮扫描电镜观察

为了解人前庭器官的超微结构，借助扫描电镜技术观察了５人（１０耳）的前庭感觉上皮。结果显示：①球囊斑呈“Ｌ”形，椭圆囊斑形如打开的贝壳；②Ｉ型感觉细胞呈烧瓶样，Ⅱ型感觉细胞呈试管状；③感觉细胞纤毛排列呈阶梯状，动纤毛最长，且位于最长的静纤毛一侧，椭圆囊斑最长的静纤毛和动纤毛造近微纹，其极性也向微纹，球囊斑则背离微纹，水平半规管壶腹嵴的动纤毛朝向椭圆囊侧，上、后半规管过来腹嵴则朝向半规管侧；④耳右形状     

豚鼠全内耳膜迷路取材术

本文介绍一种能完整取出同一内耳基底膜、血管纹、球囊斑、椭圆囊斑和膜半规管的取材方法,此法便于同时对耳蜗和前庭终器进行光镜和电镜观察。     

内耳超微结构(续十三)

某些因素引起的内耳结构变化一、氨基糖苷类抗生素引起的内耳结构改变:(一)Harada 曾观察了氨基糖苷类抗生素药物对内耳前庭器官形态结构的损害。其结果,前庭器官感觉上皮受损严重。半规管壶腹嵴,特别是其中央部分感觉上皮变性,数月后减少。感觉毛融合直至消失,少数动物整个嵴上感觉毛可完全消失。椭圆囊斑可以出现同样的变化。鼓室内注射庆大霉素后二个月,嵴的感觉毛全部消失,嵴呈现皱缩。仅在嵴周缘部分感觉毛的表面有小泡样变化,纤毛的改变从变性到消失。在椭圆囊斑也有同样变化。腹膜腔内注射硫酸链霉素后其感觉毛的变化不及鼓室内注射快,二周组仅出现感觉毛稍微变化。4周组则壶腹嵴中央部分改变,感觉毛融合,     

豚鼠内耳前庭上皮发育过程IGF—1及ERK的表达及其意义

目的：检测胰岛素样生长因子－1（insulin-like growth factor-1,IGF-1)及其受体（insulin-like growth factor-1 receptor,IGF-1R)在豚鼠前庭上皮发育过程的表达和分布特点，胞外信号调控激酶1及2（extracellular signal regulated kinase,ERK1 and 2)的活性变化，并探讨其生物学特性及意义。方法：利用免疫组织化学方法，以IGF－1和IGF－1R抗体为标记物，检测发育期豚鼠前庭器中的表达情况及分而分布特点；利用Western blot方法检测ERK1及ERK2的活性变化。结果：IGF－1及其受体可在豚鼠前庭上皮细胞中，随年龄增加而逐渐减少，ERK1及ERK2的活性随年龄的增长而逐渐降低，且ERK2的活性高于ERK1。结论：豚鼠正常发育过程内耳前庭上皮中有IGF－1及其受体的表达，为内源性的IGF－1，可能通过旁分泌和自分泌方式在局部发挥其功能，壶腹嵴感觉上皮IGF－1及其受体表达，ERK1及ERK2的活性变化与细胞增殖相同步。     

豚鼠发育过程中前庭上皮IGF—1及IGF—1R的表达变化

目的 检测胰岛素样生长因子－1（insulin-like growth factor-1,IGF-1）及其受体（insulin-like growth factor-1 receptor,IGF-1R）在豚鼠发育过程中前庭上皮的表达和分布特点,并探讨其生物学特性及意义。方法 利用免疫组织化学的方法,以IGF－1和IGF－1R抗体为标记物,检测发育期豚鼠前庭器中的表达情况及分布特点,以增殖细胞核抗原（proliferation cell nuclear antigen,PCNA0检测细胞增殖变化。结果 IGF－1及IGF－1R在鼠前诞上皮细胞中表达,随年龄增加而逐渐减少;PCNA表达于支持细胞,随年龄增加,阳性细胞数逐渐减少。结论 豚鼠正常发育过程中前庭上皮中有IGF－1及IGF－1R的表达,为内源性的IGF－1通过旁分汔和自分泌方式在局部发挥其功能,壶腹嵴感觉上皮IGF－1及IGF－1R表达与细胞增殖相同步,IGF－1在发育早期是强烈的有丝分裂原,促进细胞增殖、分化。     

核转录因子κ-Bp65在中耳胆脂瘤上皮中的表达和意义

目的检测核转录因子-κBp65（nuclear factor kappa Bp65,NF-κBp65）在中耳胆脂瘤上皮中的表达和活化,探讨其在中耳胆脂瘤发病机制中的可能作用。方法采用免疫组织化学SP法检测30例中耳胆脂瘤组织标本与15例正常外耳道皮肤标本中NF-κBp65蛋白的表达。结果NF-κBp65蛋白阳性表达定位于上皮细胞核。NF-κBp65蛋白在中耳胆脂瘤上皮组织中阳性表达率为63.3%,明显高于正常外耳道皮肤组的20.0%（P〈0.01）。结论NF-κBp65蛋白在中耳胆脂瘤上皮的异常表达可能在胆脂瘤的发生、发展过程中起重要作用。胆脂瘤上皮中NF-κBp65的活化可能参与了胆脂瘤上皮细胞过度增殖机制。     

Role of Bcl-xL protein in differentiation and apoptosis of human middle ear cholesteatoma epithelium.

OBJECTIVE: To compare the mechanisms of proliferation, differentiation, and apoptosis in middle ear cholesteatoma epithelium with those of normal external ear canal epithelium. DESIGN: The localizations of the expression of Bcl-xL protein and involucrin and the presence of apoptotic cells were determined for tissue slices of middle ear cholesteatoma epithelium and compared with the findings for normal external ear canal epithelium. In addition, SCC-25/bcl-xL transfectants showing the overexpression of Bcl-xL were used to investigate the effect of this protein on the expression of involucrin, which is a marker of epithelial cell differentiation. MATERIALS: Cholesteatoma tissue specimens were surgically excised from 10 patients. Normal skin specimens collected from the external ear canal of the 10 patients were used as control specimens. RESULTS: The expression of Bcl-xL was detected in the vicinity of the basal cell layer of both the cholesteatoma epithelium and the normal external ear canal epithelium. Conversely, the expression of involucrin (ie, a marker of epithelial cell differentiation) increased in proportion to the shallowness of the epithelial layer. In situ labeling detected apoptotic cells in the spinous and granular cell layers of cholesteatoma tissue sections and similar findings in the normal external skin specimens. Western blot analysis confirmed that the expression of involucrin protein was the same in both wild-type SCC-25 cells and the SCC-25/bcl-xL transfectants. CONCLUSIONS: In both the cholesteatoma epithelium and the normal external ear canal epithelium, differentiation and apoptosis begin when the epithelial cells separate from the basal cells. The mechanisms behind these changes, at least in apoptosis, appear to be controlled by the expression of the Bcl-xL protein.     

实验性自身免疫性内耳病耳蜗热休克蛋白70的表达

目的　探讨自身免疫性内耳病模型动物耳蜗热休克蛋白的表达。方法　利用同种异体内耳抗原免疫豚鼠 ,以建立自身免疫性内耳病 (autoimmuneinnereardisease ,AIED)动物模型 ,并应用免疫组织化学及原位杂位技术 ,研究热休克蛋白 (heatshockprotein ,hsp) 70在正常对照组及AIED模型动物实验组耳蜗中的表达情况。结果　对照组豚鼠螺旋神经节细胞中存在hsp70样蛋白基础表达 ;实验组 2 8耳中 10耳 (35 7% )听阈提高≥ 10dB ;此组动物螺旋神经节细胞和血管纹、螺旋韧带hsp70及其mRNA表达明显增强。结论　以粗制膜迷路抗原免疫豚鼠 ,听阈提高动物hsp70在螺旋神经节细胞和血管纹、螺旋韧带合成增加。     

Immunohistochemical study of cytokeratin expression in normal and pathologic middle ear mucosa of the rat.

The expression of cytokeratins in the epithelium of the middle ear and external auditory meatus of the rat was studied on cryosections of ethylenediaminetetraacetic acid-decalcified specimens by use of a panel of monoclonal antibodies. The normal middle ear epithelium revealed a complex cytokeratin profile, including regional differences. The induction of sterile middle ear effusions resulted in increased cytokeratin expression. Infective effusions were accompanied by both quantitative and qualitative changes in the cytokeratin expression patterns. The differences observed between the cytokeratin profiles of external meatal skin and those of middle ear epithelium may form a useful tool for research into cholesteatoma development.     

Immunogold TEM of otoconin 90 and otolin - relevance to mineralization of otoconia, and pathogenesis of benign positional vertigo

Implementation of the deep-etch technique enabled unprecedented definition of substructural elements of otoconia, including the fibrillar meshwork of the inner core with its globular attachments. Subsequently the effects of the principal soluble otoconial protein, otoconin 90, upon calcite crystal growth in?vitro were determined, including an increased rate of nucleation, inhibition of growth kinetics and significant morphologic changes. The logical next step, ultrastructural localization of otoconin 90, by means of immunogold TEM in young mature mice, demonstrated a high density of gold particles in the inner core in spite of a relatively low level of mineralization. Here gold particles are typically arranged in oval patterns implying that otoconin 90 is attached to a scaffold consisting of the hexagonal fibrillar meshwork, characteristic of otolin. The level of mineralization is much higher in the outer cortex where mineralized fiber bundles are arranged parallel to the surface. Following decalcification, gold particles, as well as matrix fibrils, presumed to consist of a linear structural phenotype of otolin, are aligned in identical direction, suggesting that they serve as scaffold to guide mineralization mediated by otoconin 90. In the faceted tips, the level of mineralization is highest, even though the density of gold particles is relatively low, conceivably due to the displacement by the dense mineral phase. TEM shows that individual crystallites assemble into iso-oriented columns. Columns are arranged in parallel lamellae which convert into mineralized blocks for hierarchical assembly into the complex otoconial mosaic. Another set of experiments based on immunogold TEM in young mice demonstrates that the fibrils interconnecting otoconia consist of the short chain collagen otolin. By two years of age the superficial layer of mouse otoconia (corresponding to mid-life human) has become demineralized resulting in weakening or loss of anchoring of the fibrils interconnecting otoconia. Consequently, otoconia detached from each other may be released into the endolymphatic space by minor mechanical disturbances. In humans, benign positional vertigo (BPV) is believed to result from translocation of otoconia from the endolymphatic space into the semi-circular canals rendering their receptors susceptible to stimulation by gravity causing severe attacks of vertigo. The combinations of these observations in humans, together with the presented animal experiments, provide a tentative pathogenetic basis of the early stage of BPV.     

Use of lyophilized dura in aural surgery

Lyophilized dura was utilized in chronic ear surgery to cover raw promontory surfaces, to stabilize the reconstructed ossicles, to raise the posterior insertion point of the grafted drum after canal wall removal, and to fortify the soft posterior canal wall. In 3 guinea pigs, lyodura was also placed in the bulla and studied 6, 12 and 16 months later. In 6 months of time, lyodura had become covered with normal epithelium and in 12 months of time, the greatest part of the lyodura was resorbed from the middle ear, normal epithelium having developed. In the canal wall area, resorption is slower and collagenic dural network is clearly recognizable even after a period of 24 months. Lyophilized dura contains no irritating material and is considered to be a versatile and useful material in chronic ear surgery.     

实验性急性中耳细菌感染诱发的热休克反应

目的 探讨细菌抗原和内耳抗原之间的联系及热休克反应对其抗原的影响。方法 用抗哺乳类热休克蛋白（ｂｅａｔ ｓｈｏｃｋ ｐｒｏｔｅｉｎ，ｈｓｐ）７０抗体识别绿脓杆菌、变形杆菌、大肠杆菌、金黄色葡萄球菌和肺炎克雷白杆菌抗原，进行免疫转印分析，测试正常豚鼠膜迷路中ｈｓｐ７０的表达水平。用肺炎克雷白杆菌制作急性中耳感染动物模型，观察细菌的内耳与哺乳类ｈｓｐ７０家族，正常豚鼠膜迷路表达ｈｓｐ７０水平极低。肺炎     

Expression of heat shock protein 70 in immune response of the guinea pig inner ear]

OBJECTIVE: To explore whether the immune response of the inner ear could induce heat shock protein (hsp) 70 in guinea pig cochlea. METHODS: A model of autoimmune inner ear disease (AIED) was established by systemically immunizing the guinea pig with the homologous crude inner ear antigen (CIEAg). The immunized cochleae and normal control cochleae were examined for the expression of hsp70 with techniques of immunohistochemistry and in situ hybridization. RESULTS: In the control animals, the expression of the hsp70-like protein appeared only in the spiral ganglion, whereas in the cochleae with CIEAg immunization, strong expression of the hsp70-like protein and its mRNA appeared in the spiral ganglion as well as in the stria vascularis and the spiral ligament. The hearing thresholds were significantly increased in 10 out of 28 cochleae (35.7%) with CIEAg immunization. CONCLUSION: The results suggest that the immune response of the inner ear can induce the expression of hsp70 in the guinea pig cochlea.