ABAD、雌激素受体、bcl-2、Bax在小鼠内耳中的表达差异

目的 探讨β淀粉样蛋白醇脱氢酶(amyloid β binding alcohol dehydrogenase,ABAD)、雌激素受体(estrogen receptors,Ers)、bcl-2、Bax(Bcl-associated X protein,Bcl相关X蛋白)在不同龄小鼠内耳中表达量上是否存在线性变化趋势.方法 将昆明小鼠分为2周龄、5月龄、1年三组,每组10只,采用免疫组织化学方法检测ABAD、Ers、bcl-2、Bax在不同年龄组小鼠内耳中的表达情况.结果 ABAD、Ers、bcl-2、Bax在不同龄小鼠内耳螺旋神经节细胞中均有表达,且随着年龄增大,ABAD、Bax表达逐渐增多,Ers、bcl-2表达逐渐减少.均具有统计学意义(P<0.05).结论 ABAD随年龄增大表达有增多的趋势,可能参与了内耳细胞的老化机制,可能与老年性耳聋的发生有关.     

小鼠内耳发育过程中Evi-1基因的表达

目的观察在小鼠内耳发育过程中不同阶段Evi-1基因的动态表达特征及变化规律,为进一步探索EVI-1基因在小鼠内耳发育中作用。方法选用胚胎第9.5天(E9.5)到胚胎期18.5天(E18.5)的C57BL/6胎鼠,E9.5-E15.5取胚胎头,E15.5后取内耳,之后进行冰冻切片及免疫荧光染色,利用荧光显微镜观察小鼠内耳发育过程中EVI-1基因的表达变化情况。结果在E9.5天时尚未在听泡及其周围组织观察到EVI-1基因的表达,当胚胎发育到E10.5天时,EVI-1开始在感觉上皮周围弱表达,其局限表达于听泡周围的间充质;E11.5时期Evi-1的表达增强但也局限于内耳周围的间充质;E12.5时期时感觉上皮中开始出现Evi-1的表达,但随后即消失,仅在这一时期的感觉上皮中一过性表达,E13.5后,Evi-1表达逐渐减弱至消失。结论 Evi-1基因在小鼠内耳发育时期的表达具有明显时限性以及区域性,可能与内耳感觉细胞的增殖分化有关。     

小鼠内耳毛细胞基因及其功能

近年分子遗传学和遗传工程的飞速发展，不仅加速确定了许多内耳感觉毛细胞基因，而且为进一步明确这些基因的功能，阐明它们在毛细胞分化、生长和成熟过程中的作用产生了重要的影响。小鼠转基因和通过胚胎干细胞技术的基因敲除，分别揭示了一些毛细胞基因存在基因编码的特异性启动子和它们的功能。本从毛细胞发育和成熟的不同阶段．对部分内耳毛细胞基因的表达和功能进行了概述。     

阿米卡星对小鼠内耳水通道蛋白4表达的影响北大核心CSCD

目的观察阿米卡星对小鼠内耳水通道蛋白4(aquaporin 4,AQP4)表达的影响,探讨AQP4在阿米卡星耳毒性机制中的可能作用。方法 60只CBA/CaJ小鼠随机分为实验组和对照组,每组30只,实验组小鼠皮下注射阿米卡星450mg·kg-1·d-1,每天1次,共14d,对照组小鼠皮下注射等量生理盐水。注射4天后应用免疫组化染色分别检测两组小鼠耳蜗AQP4的表达及定位情况,应用蛋白免疫印迹技术及RT-PCR技术检测两组小鼠内耳AQP4表达水平的变化。结果 AQP4在小鼠内耳表达于Corti器的支持细胞。对照组和实验组的AQP4蛋白相对含量分别为0.672±0.074、0.479±0.108,AQP4mRNA相对含量分别为0.701±0.107、0.460±0.080,实验组AQP4的蛋白及mRNA的相对含量均低于对照组,差异有统计学意义(P<0.01)。结论阿米卡星可导致小鼠内耳AQP4蛋白及基因表达的下调,这可能是阿米卡星耳毒性的机制之一。     

小鼠内耳发育的分子生物学研究进展

听基板至听泡再发育成成熟内耳的过程是一个极其复杂的过程,包括外胚层上皮的引导,听囊的初步形成和改建,感觉上皮、非感觉上皮及神经元细胞的分化等过程,最终形成精细的迷路。其间多种基因的时间、空间表达精细、准确地控制调节着这一过程。这些基因通过编码转录因子、生长因子、分泌因子或受体蛋白等重要的生物分子,以不同的机制对内耳发育起重要作用。本文综述了近年来对小鼠内耳发育的分子生物学研究状况,从内耳发育的不同阶段阐述了目前较为公认的调节小鼠内耳发育的分子生物学机制。1听基板的形成及其相关基因图1听基板的形成过程1.1…     

小鼠耳蜗胚胎发育过程中Smad4基因的表达

目的探讨小鼠内耳胚胎发育演变过程，检测Smad4基凶在小鼠耳蜗中的表达情况。方法选用耳廓反应灵敏、健康的C57BL／6小鼠作为种鼠交配，用观察阴栓方法获得适龄胎鼠，≤17天取胚胎头，≥18天在显微镜下取耳蜗，胚胎头水平冰冻切片，耳蜗平行于蜗轴冰冻切片，HE染色方法观察小鼠耳蜗发育形态演变过程，免疫组织化学方法检测Smad4蛋白在小鼠胚胎10～20天耳蜗巾的表达情况。结果胚胎10天，听泡发育．胚胎12天听泡下部有蜗管始基形成并开始发育．胚胎18天，蜗管发育2圈，形成了可以辨认的内、外毛细胞．血管纹开始分化。Smad4从胚胎15天才开始表达，最初表达部位为耳蜗底转将发育成蜗轴以及柱状上皮分化为感觉细胞及支持细胞的区域，但在胚胎早期表达较弱，后期在耳蜗内广泛表达，在螺旋器、血管纹、前庭膜均有表达，且表达逐渐增强，而在蜗轴部位的表达逐渐减弱。结论Smad4在小鼠内耳分化期有阳性表达，且表达具有明显的阶段性和区域性，说明其参与内耳听觉器官的发育过程并且可能存耳蜗的形成过程中起着重要的作用。     

17β-羟基类固醇脱氢酶10在小鼠内耳的表达与意义

17β-羟基类固醇脱氢酶10(17β-HSD10),又称短链-3-羟酰辅酶A脱氢酶(SCHAD),是位于细胞线粒体的多功能酶,参与能量代谢及激素的代谢等,在大脑的发育和老     

凋亡及其相关基因在实验性自身免疫性内耳病小鼠内耳组织中的表达

目的探讨凋亡及其相关基因在自身免疫性内耳病形成和发展中的作用。方法选用近交系C57BL／6小鼠随机数字表法分为正常对照组和免疫7、14、21、28d组，每组16只。提取豚鼠内耳膜迷路组织为抗原，与等量完令弗氏佐剂，百日咳杆菌一次免疫实验组动物，制备自身免疫性内耳病动物模型，应用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术（terminal deoxynucleotidyl transferase—mediated d-UTP nick end—labing，TUNEL）检测内耳中的细胞凋亡，应用免疫组化和逆转录聚合酶链反应（RT—PCR）技术检测Fas、FasL及bcl-2在内耳的表达。结果正常小鼠内耳组织中，TUNEL染色阳性细胞极为少见，偶尔在Corti器或球囊斑的支持细胞发现。免疫7d后，内毛细胞和少量的血管纹边缘细胞TUNEL染色阳性，14d后TUNEL染色阳性的细胞数量及种类显著增加，但外毛细胞、螺旋神经节细胞与前庭神经节细胞免疫前后均未见凋亡表达。免疫组化染色显示，正常小鼠内耳中Fas表达广泛，FasL存部分螺旋神经节细胞与前庭神经节细胞表达，bcl-2仅在螺旋神经节细胞、前庭神经节细胞有较强表达。免疫后FasL在各种组织均有较强表达，bcl一2在外毛细胞出现表达，在耳蜗神经无细胞的表达增加。RT—PCR检测正常小鼠内耳组织的Fas mRNA、FasL mRNA、bcl-2mRNA均为阳性，FasL mRNA低水平表达，免疫后升高，在2周达到高峰后逐渐下降；bcl-2 mRNA在免疫后进行性升高。结论Fas／FasL信号系统介导的凋亡与自身免疫性内耳病的发生、发展过程关系密切，bcl-2对内耳中Fas／FasL介导的凋亡有重要的调节作用。     

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。     

小鼠内耳COCH基因的表达

目的观察小鼠内耳COCH基因的表达。方法体外转录合成地高辛标记的反义RNA探针,内耳样本冰冻切片,进行原位杂交反应。结果发现除已报道的耳蜗螺旋韧带、壶腹嵴间质等内耳组织外,COCH基因在内淋巴囊周围间质成纤维细胞亦表达阳性,其mRNA水平(原位杂交染色平均积分光密度0.68±0.25)与耳蜗螺旋韧带(0.77±0.19)相似,强于壶腹嵴间质(0.47±0.21)。结论COCH基因在小鼠内淋巴囊周围间质细胞高表达,为COCH基因内耳功能的后续研究及其与梅尼埃病关系的探究提供了线索。     

正常豚鼠内耳水通道蛋白的表达及意义

目的：检测正常豚鼠内耳组织中水通道蛋白（aquaporins,AQPs）的表达,探讨其在内耳液体平衡中的意义.方法：用免疫组织化学方法,以兔抗大鼠AQP0、1、2、3、5、7、8的多克隆抗体,检测正常豚鼠内耳组织中水通道蛋白亚型0、1、2、3、5、7、8的表达.结果：水通道蛋白亚型0、1、2、3、5、7、8在豚鼠内耳有不同程度、不同模式的表达,其中AQP0仅在血管纹上皮细胞、螺旋神经节细胞有较弱的表达,AQP1的分布见于包绕骨迷路、内淋巴囊、内淋巴管的纤维细胞,基底膜鼓阶面细胞、螺旋韧带纤维细胞、螺旋缘纤维细胞、Corti器、内外螺旋沟、血管纹、椭圆囊壁、球囊壁、螺旋神经节细胞等.AQP2表达在血管纹、Corti器、螺旋神经节细胞和内淋巴囊中.AQP3、7、8的分布类似,在螺旋神经节和包绕膜迷路的组织中均有表达,其中Corti器、内外螺旋沟、血管纹、螺旋神经节表达较强,在螺旋韧带、螺旋缘纤维细胞表达较弱.AQP5则在Corti器、内外螺旋沟、螺旋神经节细胞表达较强,在螺旋韧带纤维细胞表达稍弱.结论：在正常豚鼠内耳中,尤其是膜迷路中有多种水通道蛋白亚型,以不同的方式表达,他们可能在维持膜迷路液体平衡中起着协同作用.     

Expression of glycoconjugates in the mouse inner ear after prenatal irradiation

Summary Irradiation of the murine fetal inner ear is known to produce damage both to the vestibular and cochlear parts in the adult mouse. Fluorescein-labelled lectins were used to reveal possible differences in the glycoconjugate content between normal and irradiated inner ears. In the vestibular part, the otoconia showed the highest uptake of labelled sugars. This uptake was weaker after irradiation when compared to non-irradiated specimens. The type I hair cells in the ampulla and in the utricle showed a weaker uptake, but no labelling was demonstrated in the type II hair cells compared to the non-irradiated controls. In the cochlear part of the inner ear almost no uptake of fluorescent-binding lectins could be demonstrated in the irradiated groups except for in the tectorial membrane. In the endolymphatic sac no uptake was shown after prenatal irradiation. These findings are discussed and correlated to the already known damage of the inner ear following prenatal irradiation.     

Expression of S-100 protein in the human fetal inner ear

The expression of S-100 protein was analyzed in the human fetal inner ear using immunohistochemical methods. In the 11-week-old human fetus, the cochlea was almost negative for S-100 protein, whereas in the 14- and 15-week-old fetuses, the spiral ligament, Reissner's membrane and spiral limbus were positive for the protein. These results suggest that S-100 protein may be a reliable marker for determining functional maturation of the fetal cochlea and the inner ear. In the l l-, 14- and 15-week fetuses, the epithelial cells of the endolymphatic sac were labelled with S-100 protein. These findings demonstrate that the endolymphatic sac, spiral limbus and spiral ligament in the fetal inner ear have a high activity of S-100 protein, with this presence possibly related to fluid and ion transport of endolymph.     

浆膜蛋白RTN1和RTN4基因在小鼠内耳的表达

目的 探讨浆膜蛋白RTN1和RTN4基因在小鼠内耳的表达。方法 采用5只成年小鼠内耳组织提取总RNA，逆转录后获得小鼠内耳细胞cDNA，根据RTN1和RTN4基因编码区序列设计的引物进行PCR扩增，通过PCR产物分析和DNA测序确定RTN1和RTN4是否在小鼠内耳细胞表达。结果 采用小鼠内耳组织总RNA，RT—PCR扩增出RTN1和RTN4基因部分编码区，扩增产物测序证实小鼠内耳中有RTN1和RTN4基因的表达。结论 RTN1和RTN4基因在内耳有表达，为RTN1和RTN4与连接蛋白26（connexin26）蛋白的互作关系提供了进一步的证据。浆膜蛋白RTN1和RTN4可能与连接蛋白26在听觉生理中起作用。     

Age effects and size effects in the ears of gekkonomorph lizards: inner ear

Audiograms have indicated greater auditory sensitivity in larger than in smaller geckos; part of this difference, interspecifically and intraspecifically, is explained by middle-ear proportions. To investigate the contribution of the inner ear to the variation in sensitivity, we examined it in museum specimens representing 11 species and three subfamilies. We measured papilla basilaris length, and, when intact, the saccular otoconial mass. Papilla length approximated 1% of rostrum-anus length in large geckos but 2% in small geckos; in some species some inter-aural difference was indicated. Over the lumped material, relative papilla length varied as a function of body length, with highly significant correlation. Similar relations prevailed within each subfamily. However, intraspecifically the correlation of papilla basilaris length with animal size was usually nonsignificant. Hair cell populations assessed from SEM photographs were larger in the larger species but intraspecifically did not relate to an individual’s size. Hence interspecifically, the dependence of auditory sensitivity on animal size seems supported by inner-ear differences but intraspecifically this relation derives only from the middle ear. Otoconial mass, as measured by its volume, was correlated with animal length both interspecifically and intraspecifically.     

不同内耳组织抗原免疫致自身免疫性感音神经性聋的研究

目的：探讨不同内耳组织抗原免疫所致内耳主要病理损伤部位和听力障碍类型。方法：采用同种螺旋韧带（ＳＬ）、基底膜（ＢＭ）、螺旋神经节（ＳＧ）组织抗原免疫豚鼠，观察内耳组织病理改变和听觉功能变化。结果：ＳＬＡｇ和ＢＭＡｇ免疫组主要表现耳蜗微音器电位阈值升高和复聪现象，以及蜗管内和血管纹的免疫炎性病理改变；ＳＧＡｇ免疫组主要表现听神经复合动作电位阈值升高和幅值降低，内耳病理变化主要位于蜗轴血管及周围和ＳＧ     

Cochlear spiral ganglion cell degeneration in wild-caught mice as a function of age

Presbyacusis in humans is an age-related bilateral sensorineural hearing impairment generally associated with degeneration of cochlear hair cells and spiral ganglion cells (SGC) predominantly in the basal turn but present in the apical turn. Investigations of cochleas of aged rats and gerbils reveal a large loss of SGCs in the apical as well as the basal turns. Genetically inbred aged mice, on the other hand, seem to have variable amounts of SGC loss beginning in some strains very early in the life span of the animals and greatest in the basal turn.

Theee age groups of wild-caught, then laboratory-bred, mice were investigated to determine the pattern of SGC degeneration. In 18–19-month-old animals the main loss of SGCs occurred in the basal turn (49% loss compared to 2–3 months) followed by the apical turn (31%). The greatest SGC losses in the 28–31-month-old animals were in both the apical (76%) and basal turns (74%). Thus, this strain of mice is similar to other rodents in that both ends of the ganglion are affected by SGC degeneration associated with aging.     

两种不同细菌所致急性中耳炎对内耳损伤的差异

目的研究两种毒力不同的细菌导致的急性中耳炎对内耳超微结构损伤的差异.方法豚鼠麻醉后,一组在无菌条件下于右耳经鼓膜注射1×108·L-1的绿脓杆菌液100μL,左耳作正常对照;另一组同法注射金葡菌液.三天后常规处理,透射、扫描电镜观察.结果造模后三天,绿脓杆菌感染组及金葡菌感染组基底膜、血管纹均可见细胞损伤、细胞器变性,绿脓杆菌感染组内耳超微结构的改变明显重于金葡菌感染组.结论急性中耳炎早期即可有内耳的形态学改变,绿脓杆菌引起的急性化脓性中耳炎可能会导致严重感音神经性聋.     

Effect of lipooligosaccharide mutations of Haemophilus influenzae on the middle and inner ears

Objective

The purpose of this study was to determine the virulence of nontypeable Haemophilus influenzae 2019 (NTHi 2019) and its two lipooligosaccharide (LOS) mutant strains, B29 (gene htrB) and DK1 (gene rfaD), and compare their effect on the middle ear, round window membrane, and inner ear.

Results

Fifteen chinchillas were divided into three equal groups and their bullas inoculated bilaterally with 0.5 ml of 10² CFU/ml of parent NTHi 2019, B29 or DK1 mutant strains. Two days after inoculation all animals had otitis media and inflamed middle ear mucosa. There was a trend of greater thickness and infiltration of the round window membrane in animals inoculated with the wild-type NTHi strain compared to the mutant strains and a significant increase in both inflammatory cell infiltration and bacteria presence in the scala tympani area of the inner ear. Strial edema was only observed in the wild-type-inoculated group.

Conclusions

LOS mutants of NTHi appear to have a reduced ability to pass through the round window membrane resulting in less inner ear inflammation and pathological changes.