豚鼠椭圆囊斑微纹区和边缘区毛细胞损伤后恢复的比较观察

利用扫描电镜和连续半薄切片的观察，对豚鼠椭圆囊斑微纹区和边缘区的毛细胞损伤后的恢复情况进行研究。结果，正常豚鼠的椭圆囊斑微纹区的感觉细胞主要是Ⅰ型毛细胞，连续１２天皮下注射庆大霉素（１００ｍｇ／ｋｇ），豚鼠椭圆囊斑的损伤主要发生在微纹区，损伤后较长存活期实验组豚鼠的椭圆囊斑微纹区中，形态类似Ⅱ型毛细胞的感觉细胞占绝大多数。提示这些类似Ⅱ型毛细胞的感觉细胞是修复过程中产生的毛细胞。     

豚鼠椭圆囊斑微纹区和边缘区毛细胞损伤后恢复的比较观察

利用扫描电镜和加续半薄切片的观察，对豚鼠椭圆囊斑微纹区和边缘区的毛细胞损伤后的恢复情况进行研究，结果，正常豚鼠的椭圆囊斑微纹区的感觉细胞主要是Ｉ型毛细胞，连续１２天皮下注射庆大霉素，豚鼠椭圆囊斑的损伤主要发生在微纹区，损伤后较长存活期实验组豚鼠的椭圆囊斑微纹区中，形态类似Ⅱ型毛细胞的感觉细胞占大多数，提示这些类似Ⅱ型毛细胞的感觉细胞是修复过程中产生的毛细胞。     

庆大霉素耳中毒后豚鼠椭圆囊斑和耳蜗毛细胞损伤与再生的观察

庆大霉素耳中毒后豚鼠椭圆囊斑和耳蜗毛细胞损伤与再生的观察刘冰李西秦吴润身王锦玲刘顺利哺乳类动物内耳毛细胞损伤后能否再生和修复受到广大学者的关注。我们采用扫描、透射电镜和听性脑干反应（ＡＢＲ）对豚鼠椭圆囊斑和耳蜗毛细胞在庆大霉素损伤后的再生和修复能力进...     

豚鼠前庭椭圆囊器官在庆大霉素损伤后离体培养的细胞增殖

目的 在离体状态下对豚鼠前庭椭圆囊器官进行培养,观察庆大霉素损伤毛细胞后细胞的增殖情况。方法采用白色健康豚鼠36只,体重300-450克,随机分为4组:(1)正常对照组;(2)正常加BrdU(5-溴基脱氧尿核苷BrdU)组;(3)庆大霉素组;(4)庆大霉素加BrdU组。应用体外组织培养、5—溴基脱氧尿核苷(BrdU,3μg/ml)掺入及免疫组织化学染色和Epon树酯包埋半薄切片等方法,在不同时间(2、3、5、15天)光镜观察庆大霉素(2.0mM)损伤后毛细胞及支持细胞增殖情况。结果 用庆大霉素48小时后,可见到椭圆囊感觉上皮中的毛细胞溶解破坏。加入BrdU继续培养,BrdU免疫组织化学染色显示了第5和15天被BrdU标记的阳性细胞核,阳性细胞核多位于椭圆囊感觉上皮中基底层,从细胞的形态和分布位置上看为支持细胞;感觉上皮的表层,也可观察到BrdU反应阳性的上皮细胞,细胞核较圆,形态和位置类似毛细胞。结论 提示损伤后的椭圆囊感觉上皮支持细胞可以进行细胞的有丝分裂,支持细胞可能是修复毛细胞的前体细胞。     

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

981929表皮生长因子对内耳前庭感觉上皮再生的促进作用/崔鹏程…声解放军医学杂志一19于)8 .23(l)一28 为进一步研究促内耳前庭毛细胞再生的因索,用器官培养技术结合放射自显影.观察表皮尘长因子(E〔万)对小鼠内耳前庭毛细胞和支持细胞再生的影响3。个椭圆囊斑分成实验组和对照组经庆大霉素破坏毛细胞和支持细胞后,在实验组中力lJ入IC(拼和,H一ljIR培养9天后制作2脚11半薄切片。放射自显影15天.光镜观察结果发现:各组中毛细胞均未受到标记。支持细胞受到标记且王要集中在边缘区。‘灾验组中平均每张切片中受标记的支持细胞数为5.1士1.8,…     

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…     

磷酸化JNK在庆大霉素致豚鼠前庭毛细胞凋亡中的表达

目的:观察庆大霉素致豚鼠前庭毛细胞损伤特点,并探讨磷酸化cjun氨基末端激酶(JNK)在前庭毛细胞凋亡过程中的作用机制。方法:健康豚鼠随机分为实验组和对照组各15只,分别行100mg/(kg·d)庆大霉素和生理盐水连续肌肉注射7d,第8天全身麻醉下处死豚鼠,取壶腹嵴为观察对象,左耳铺片,硝酸银染色检测毛细胞损伤;右耳冷冻连续切片,DNA末端转移酶介导的缺口末端标记法(TUNEL)检测毛细胞凋亡,免疫组织化学方法检测细胞中JNK磷酸化。结果:实验组中毛细胞受损,壶腹嵴顶端见凋亡细胞,并在细胞内观察到磷酸化JNK免疫阳性反应;对照组中未见毛细胞损伤及凋亡细胞,未见磷酸化JNK。结论:庆大霉素可以通过诱导凋亡造成内耳前庭毛细胞损伤,同时磷酸化的JNK标志着JNK途径的活化,提示JNK途径在细胞凋亡过程中发挥了一定作用。     

声诱发短潜伏期负电位豚鼠的内耳铺片研究

目的 在耳毒性损伤的豚鼠模型上诱发声诱发短潜伏期负电位( acoustically evoked short latency negative response,ASNR),通过内耳铺片观察ASNR豚鼠的基底膜球囊、椭圆囊及半规管壶腹的组织形态学特点,验证豚鼠ASNR的责任终器.方法 将45只健康豚鼠按随机数字表法分为2组,健康对照组15只(30耳),药物致聋组30只(60耳).致聋组给药(硫酸卡那霉素+利尿酸)致聋7～10d后,行听觉脑干反应(ABR)测试,根据ASNR引出情况进一步分为ASNR组和非ASNR组.三组豚鼠断头取颞骨,解剖显微镜下取出基底膜、球囊斑、椭圆囊斑和壶腹嵴,通过显微镜观察毛细胞数目和形态变化.结果 致聋组有27只动物(54耳)完成测试,其中45耳达到重度感音神经性聋,19耳引出ASNR(35.2％),阈值为110～125 dBSPL,平均阈值(121.7±4.5)dBSPL,潜伏期1.80～2.08 ns,平均潜伏期(1.93±0.07)ms.铺片观察显示,基底膜、球囊、随圆囊、壶腹嵴毛细胞密度按正常对照组、ASNR组、非ASNR组依次减低,毛细胞损伤程度依次加重.ASNR组球囊微纹区、周边区毛细胞密度与对照组差异无统计学意义(P值均＞0.05);其他各组的相应比较,差异均有统计学意义(P值均＜0.05).结论 豚鼠ASNR的责任终器是球囊,而不依赖于耳蜗、椭圆囊及半规管功能.     

庆大霉素鼓室内注射后在内耳细胞中的分布

目的 观察鼓室内注射庆大霉素后，不同时间庆大霉素在前庭和耳蜗中的分布。方法 将庆大霉素同德州红连接形成庆大霉素-德州红耦联物后，行豚鼠鼓室内注射，注射后12h，1、2、3、4、7、14、28d处死动物，Phalloidin染色后运用激光共聚焦扫描显微镜观察基底膜、椭圆囊、球囊、外半规管壶腹嵴庆大霉素分布情况，并进行荧光分布半定量分析。结果 庆大霉素自注射后12h起在内耳所有细胞均见分布，在基底膜的外毛细胞、椭圆囊、球囊、外半规管壶腹嵴的感觉细胞聚集明显，主要聚集在毛细胞顶端纤毛下方的细胞质中，支持细胞分布较少。注射后第3天庆大霉素在内耳聚集达到最高峰，并在毛细胞内聚集较长时间。结论 庆大霉素-德州红耦联物是一个研究庆大霉素在内耳分布的良好的荧光探针，可用来检测庆大霉素的药代动力学和聚集机制．     

Direct Evidence of Nitric Oxide Production in Guinea Pig Vestibular Sensory Cells

Production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. The utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, and significantly increased after inoculation with LPS. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role for the vestibular physiology and also be involved in disease of the inner ear.     

Direct evidence of nitric oxide production in guinea pig vestibular sensory cells

Production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. The utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, and significantly increased after inoculation with LPS. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role for the vestibular physiology and also be involved in disease of the inner ear.     

Organ culture of the crista ampullaris of the embryonic guinea pig

The embryonic inner ear of the guinea pig was cultured one to ten days in vitro. The explantation occurred on the 30th and 40th gestational days, respectively. Hair cells were found in various stages of cytodifferentiation. Both hair cells and supporting cells were ultrastructurally preserved at the end of the period in vitro. The gross morphologic features of the crista ampullaris were still immature. The embryonic inner ear of the guinea pig can be maintained in organ culture until at least as late as the 46th gestational day.     

Ultrastructural findings in the inner ear of Jackson shaker mice.

The ultrastructural characteristics of the inner ear of Jackson shaker mice were analyzed. We used 12 Jackson shaker mutants (js/js) with ages ranging from 10 to 47 days and 10 heterozygotes of the Jackson shaker (js/+) with ages ranging from 10 to 30 days. The most striking findings observed were incomplete differentiation of the stereocilia of the outer hair cells and the maculae, although outer and macular hair cell cytoplasm, including the nerve terminals, became fully developed. Most outer hair cells did not show regular W-shaped configuration of the stereocilia throughout the entire turns of the cochlea except for a few hair cells. In many hair cells of the utricular and saccular maculae, the classical pipe organ configuration of the stereocilia was not observed. The Jackson shaker mice have been reported to have a gene abnormality on chromosome 11, and its gene locus was close to that of our new-mutant mice which showed deranged stereocilia of the outer and macular hair cells. Therefore, future studies can provide additional information on the cytodifferentiation of the stereocilia as a function of the gene on chromosome 11.     

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.     

人类耳蜗感觉细胞自然培养方法探讨

目的在体外对人类耳蜗感觉细胞进行成功培养。方法参照用于豚鼠的有关技术,选择死亡6 h内的新生儿尸体,取出其耳蜗,采用微分离技术分离耳蜗感觉上皮细胞块并对细胞块酶解,加入可分量的细胞培养液制成含低密度的分离细胞混悬液,分置培养皿中,置培养箱中培养。结果获得了对人类耳蜗感觉细胞进行分离和培养的技术。结论成功分离出耳蜗感觉细胞并使用自制的细胞培养液进行了长期自然培养,为以后进一步研究耳蜗感觉细胞（包括耳蜗干细胞）积累了经验。     

碱性成纤维细胞生长因子对前庭毛细胞的保护作用

目的 观察碱性成纤维细胞生长因子对新霉素所致离体椭圆囊毛细胞损害的保护作用。方法 采用离体的豚鼠椭圆囊培养法，对照组为普通前庭培养液培养，实验组按培养液中含和不含ｂＦＧＦ分组，用新霉素造成各实验组２的椭圆囊毛细胞损伤，各组椭圆均行扫描电镜检查并行毛细胞计数。结果 含ｂＦＧＦ的实验组毛细胞的存活数目显著高于不含ｂＦＧＦ的实验组，对照组无缺失或损伤。结论新霉不对离体的椭圆囊毛细胞的有罗强的破坏作用，ｂ     

Glutamate-induced Production of Nitric Oxide in Guinea Pig Vestibular Sensory Cells

Glutamate-induced production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with glutamate, NMDA and AMPA. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role in the glutamate-induced ototoxicity and also be involved in disease of the inner ear.     

Glutamate-induced production of nitric oxide in guinea pig vestibular sensory cells

Glutamate-induced production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with glutamate, NMDA and AMPA. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role in the glutamate-induced ototoxicity and also be involved in disease of the inner ear.     

Spontaneous hair-cell renewal following gentamicin exposure in postnatal rat utricular explants

We have established an in vitro model of long-time culture of 4-day-old rat utricular maculae to study aminoglycoside-induced vestibular hair-cell renewal in the mammalian inner ear. The explanted maculae were cultured for up to 28 days on the surface of a membrane insert system. In an initial series of experiments utricles were exposed to 1 mM of gentamicin for 48 h and then allowed to recover in unsupplemented medium or in medium supplemented with the anti-mitotic drug aphidicolin. In a parallel control series, explants were not exposed to gentamicin. Utricles were harvested at specified time points from the second through the 28th day in vitro. Whole-mount utricles were stained with phalloidin–fluorescein isothiocyanate and their stereociliary bundles visualized and counted. In a second experimental series 2′-bromo-5′deoxyuridine labeling was used to confirm the antimitotic efficacy of aphidicolin. Loss of hair-cell stereociliary bundles was nearly complete 3 days after exposure to gentamicin, with the density of stereociliary bundles only 3–4% of their original density. Renewal of hair-cell bundles was abundant (i.e. 15× increase) in cultures in unsupplemented medium, with a peak of stereociliary bundle renewal reached after 21 days in vitro. A limited amount of hair-cell renewal also occurred in the presence of the anti-mitotic drug, aphidicolin. These results suggest that spontaneous renewal of hair-cell stereociliary bundles following gentamicin damage in utricular explants predominantly follows a pathway that includes mitotic events, but that a small portion of the hair-cell stereociliary bundle renewal does not require mitotic activity.