新生大鼠耳蜗Corti器体外培养及其转基因表达

目的建立新生大鼠耳蜗Corti器体外培养模型及观察外源基因表达。方法采用出生后1~3天龄大鼠,分离出的耳蜗Corti器组织置入表面覆盖有胶原凝胶的培养皿中,在含有10%DMEM液体培养基中培养;加入携带报告基因的腺病毒悬液,观察外源基因的表达。结果耳蜗Corti器体外培养8小时后,培养组织生长良好,形态结构清晰可辨,在该实验条件下,Corti器形态结构可以维持5天以上,最长达7天,随着培养时间的延长,由于周围组织生长的蔓延,Corti器的结构分界不清楚。腺病毒加入Corti器后12小时即有基因表达,24小时达到高峰,表达时间可持续1周。结论新生大鼠离体内耳组织转基因培养法是内耳Corti器体外培养实验研究的一种可行方法。     

庆大霉素对小鼠耳蜗毛细胞损害的离体培养试验模型

目的建立庆大霉素损坏小鼠耳蜗毛细胞的离体培养试验模型.方法应用耳蜗基底膜分离取材技术,耳蜗器官离体培养技术和毛细胞纤毛荧光染色技术定量观察了不同浓度庆大霉素对新生C57小鼠全耳蜗毛细胞的损害规律.结果庆大霉素对耳蜗毛细胞的损害程度随着剂量的增加而加重,耳蜗毛细胞的损害首先发生在外毛细胞,其病变规律是从耳蜗的底回向顶回发展.结论庆大霉素对离体培养耳蜗毛细胞的损害规律与动物活体试验的损害规律基本相同.     

重组杆状病毒载体介导新生大鼠耳蜗Corti器体外模型基因转染研究

目的建立新生大鼠耳蜗Corti器体外培养模型,探讨重组杆状病毒作为内耳基因转染载体的可行性及其转染特点。方法应用Bac-to-Bac杆状病毒表达系统制备重组杆状病毒Bac-GFP,建立新生大鼠耳蜗Corti器体外模型,加入携带报告基因的病毒悬液,观察外源基因表达情况。结果耳蜗Corti器体外培养48小时后,培养组织生长良好。Bac-GFP联合丁酸钠可以高效转染毛细胞和螺旋神经节细胞,同时并没有改变Corti器的正常结构。结论新生大鼠离体内耳组织转基因培养法是内耳基因治疗实验研究的一种可行方法。杆状病毒可以在体外高效、安全、快速的转染毛细胞和螺旋神经节细胞,有希望成为内耳基因治疗的新型载体。     

庆大霉素对离体培养小鼠前庭终器的损害

目的：观察并建立不同浓度庆大霉素损害新出生C 57小鼠前庭各终器的离体实验模型。方法：应用前庭终器分离取材技术、前庭器官离体培养技术和组织学检查技术观察不同浓度庆大霉素对新出生C 57小鼠前庭各终器的损害。结果：庆大霉素对离体培养前庭毛细胞的损害模式是随着剂量的增加而损害加重，庆大霉素对离体培养前庭毛细胞的破坏与其在体实验的耳毒性规律基本相似。结论：囊斑微纹区和壶腹嵴顶部的毛细胞比周边区的毛细胞更易遭受庆大霉素的破坏。     

模拟失重和噪声对大鼠听功能及耳蜗Corti 器损伤的协同作用

目的：探讨模拟失重和噪声对大鼠听功能及耳蜗Corti器损伤的协同作用。方法48只大鼠随机分为空白组、失重组、噪声组和失重＋噪声组,每组12只。失重组以Morey －Holton法模拟失重环境,噪声组暴露的噪声环境为模拟航天载人飞船内复合噪声,包括72±2 dB SPL的持续稳态噪声和160 dB SPL的脉冲噪声,失重＋噪声组则同时暴露于上述失重及噪声环境中,空白组常规饲养,不做任何处理。分别于暴露1周和2周后,每组随机选出6只大鼠行ABR阈值检测后取材行 HE染色、免疫荧光（DAPI）染色及扫描电镜观察耳蜗的形态学变化。结果暴露2周后大鼠ABR阈值较暴露1周时升高,失重＋噪声组ABR阈值最高（ P＜0．01）,其次为噪声组。HE染色示暴露1周和2周后除空白组外各组大鼠耳蜗Corti器均有不同程度的损伤,失重＋噪声组最严重。免疫荧光染色示暴露1周后大鼠耳蜗毛细胞死亡方式以肿胀坏死为主,失重＋噪声组最严重（肿胀率10％）,其次为噪声组（肿胀率3．33％）;暴露2周后大鼠耳蜗外毛细胞以核缺失为主,失重＋噪声组缺失率最高（缺失率13．6％）,其次为噪声组（缺失率12．7％）,失重组毛细胞缺失以内毛细胞为主。扫描电镜示,失重＋噪声组毛细胞静纤毛损伤最严重,其次为噪声组,再次为失重组。结论模拟失重和噪声环境对大鼠听功能的影响有协同作用,且这种协同作用对耳蜗Corti器有显著的损伤作用。     

强次声波对豚鼠Corti器超微结构的损伤

目的 观察强次声波对豚鼠Corti器超微结构的损伤情况。方法 将豚鼠置于频率8Hz、强度为135dBSPL的次声声场中连续暴露90min。应用扫描电镜分别观测强次声波暴露后即刻（2h内）、2天和7天时动物Corti器超微结构的变化，计算各组耳蜗的受损率，与对照组进行比较。结果 扫描电镜下见各实验组动物Corti器感觉毛细胞纤毛缺失、散乱、倒伏，表皮板等结构均有不同程度的损伤。在存活较长时期后，还可见到纤毛融合．部分细胞的表皮板破裂，以及支持细胞分离，细胞溶解，形成空洞。耳蜗受损率分别为70％～80％。结论 强次声波可引起豚鼠Corti器超微结构不同程度的损伤。     

重组腺病毒构建及转染培养耳蜗Corti器和螺旋神经节细胞的实验研究

目的构建携带绿色荧光蛋白（GFP）基因的重组腺病毒,观察GFP基因在体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞的表达。方法通过细菌内同源重组方法构建携带有绿色荧光蛋白基因的重组腺病毒（Ad-GFP）,观察重组腺病毒转染培养的新生大鼠耳蜗Corti器和螺旋神经节细胞后,不同时间内绿色荧光蛋白基因的表达情况、表达部位及病毒对培养耳蜗Corti器和螺旋神经节细胞的影响。结果构建的重组腺病毒经酶切电泳鉴定正确;荧光显微镜下观察腺病毒转染体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞后,12 h即开始表达绿色荧光蛋白基因,24 h达到高峰,表达时间可持续1周;倒置显微镜观察转染后的Corti器和螺旋神经节细胞形态结构及生长无明显改变。结论本实验成功构建了携带绿色荧光蛋白（GFP）基因的重组腺病毒,通过该方法构建的腺病毒可以介导绿色荧光蛋白（GFP）基因在体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞中表达,但腺病毒本身对体外培养的耳蜗Corti器和螺旋神经节细胞生长无明显影响,为通过腺病毒进行内耳基因治疗提供了理论依据。     

硫酸链霉素引起的大鼠体外培养耳蜗毛细胞凋亡

目的 探讨在离体培养条件下硫酸链霉素是否通过半胱氨酸天冬氨酸蛋白酶(caspase)的激活而导致大鼠耳蜗毛细胞凋亡.方法 将出生后3～4 d的大鼠耳蜗基底膜取出进行离体培养,培养过夜后用0.1 mmol/L硫酸链霉素培养液继续培养24 h.终止培养前1 h应用碳氧荧光素标记的肽荧光甲基酮(carboxyfluorescein labeled peptide fluoromethyl ketone,FAM-peptide-FMK)标记的caspase-6,caspase-8,或caspase-9指示剂作用1 h,然后应用异硫氰酸四甲基罗丹明(tetramethyl rhodamine iso-thiocyanate,TRITC)标记的鬼笔环肽(phalloidin)标记毛细胞的纤毛和表皮板,再应用Topro-3 DNA探针对细胞核进行染色,最后在共聚焦显微镜下观察.结果 1 mmol/L硫酸链霉素引起的耳蜗毛细胞缺失在底回约为80%,在顶回约为20%.经链霉素作用后,耳蜗毛细胞的细胞核发生浓缩和碎裂,同时可见cmpase-6,caspase-8和caspase-9阳性表达.结论 硫酸链霉素可以引起离体培养的大鼠耳蜗毛细胞凋亡,上游和下游的caspase激活可能是其主要途径.     

Identification of factors that maintain mammalian outer hair cells in adult organ of Corti explants

Both outer hair cells (OHCs) and inner hair cells (IHCs) survive and mature in 3 days old rat organ of Corti explants cultured for 1 month in a minimal essential medium. In contrast, under the same culture conditions, only IHCs survive in explants from adult guinea pig organ of Corti while many of the OHCs are lost within the first 48 h. Hair cell counts show OHCs loss to be greater in the lower portion (i.e. middle turn) of the cochlea than at the apex. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) indicates that there is DNA damage in adult OHCs within 8 h of explantation. Treatment of the adult organ of Corti explants with either actinomycin D (10⁻⁷ M) or cycloheximide (10⁻⁶ M) prevents most OHC losses. According to these results apoptosis may be the mechanism of OHC loss in adult organ of Corti explants. Stable membrane potentials recorded from the OHCs in both uncultured and actinomycin D-treated organ of Corti explants cultured for 72 h demonstrate the functional integrity of these hair cells. OHC losses in the adult guinea pig organ of Corti cultures can also be prevented by treatment with several of the growth factors tested, i.e. acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), and glial cell-derived neurotrophic factor (GDNF). The results of this study suggest that growth factor therapy may be applicable to the treatment of some hearing disorders.     

重组腺病毒Ad-GFP转染新生小鼠离体耳蜗基底膜的实验分析

目的观察重组腺病毒Ad-GFP转染新生小鼠离体耳蜗基底膜培养组织的情况。方法取新生1～5 d小鼠的耳蜗基底膜,离体培养1 d后,加入重组腺病毒Ad-GFP继续培养1 d,在荧光显微镜及Confocal显微镜下观察病毒对离体耳蜗基底膜的转染情况。结果耳蜗基底膜离体培养1 d后,在显微镜下观察,可见基底膜贴壁生长良好,外周有新生上皮细胞和成纤维细胞长出;高倍显微镜下可见耳蜗内外毛细胞和支持细胞等结构。离体耳蜗基底膜培养组织加入重组腺病毒Ad-GFP继续培养1 d后,可见重组腺病毒Ad-GFP能高效转染新生小鼠离体耳蜗基底膜及其外周长出的新生上皮细胞和成纤维细胞;在新生小鼠离体耳蜗基底膜上,不仅大上皮嵴细胞区域、小上皮嵴细胞区域的细胞能被高效转染,毛细胞也能被高效转染。结论重组腺病毒Ad-GFP能高效转染新生小鼠离体耳蜗基底膜培养组织。     

Cochlear permeability of neomycin and gentamicin: an immunohistochemical study

Summary Aminoglycoside antibiotics (AGAs) target specifically the cochleo-vestibular hair cells, but with varied ototoxicity. Differences in their penetration and clearance rates into the membranous labyrinth may play a role. This in turn may be related to a difference in the number of amine groups, the cationic nature, as well as the molecular weight and size of the AGA molecule. Immunohistochemical labeling techniques were used to study the pathways of gentamicin and neomycin from the perilymph into cochlear tissues and target cells. The more cochleotoxic AGA, neomycin, penetrated into cochlear tissues faster than the less cochleotoxic AGA, gentamicin. Offprint requests to: A. J. Duvall III     

体外诱导大鼠骨髓间充质干细胞向内耳毛细胞样细胞分化的研究

目的 探讨体外定向诱导大鼠骨髓间充质干细胞向耳蜗毛细胞样细胞分化的可行性.方法 分离培养大鼠骨髓间充质干细胞,并加以鉴定.分离出生1～3 d的乳鼠耳蜗Corti器,与骨髓间充质干细胞在体外共培养14 d.通过反转录聚合酶链反应(RT-PCR)和免疫细胞化学染色对分化细胞的特异性分子指标(myosinⅦa、math1及calretinin)进行鉴定.结果 免疫细胞化学染色显示诱导分化后的细胞myosinⅦa、math1和calretinin表达阳性,RT-PCR提示分化后的细胞可表达毛细胞的标志物myosinⅦa和math1.结论 体外培养的骨髓间充质干细胞可诱导分化为具有毛细胞分子标志物的毛细胞样细胞.     

Uptake of horseradish peroxidase from perilymph by cochlear hair cells

The specific mechanisms involved in the uptake of an exogenous protein, horseradish peroxidase (HRP), by cochlear hair cells were studied morphologically in light and electron microscopy. HRP was internalized by coated vesicles which formed from the plasma membrane only in the basal portion of the hair cells. Inner hair cells demonstrated relatively greater uptake than adjacent outer hair cells. The lateral plasma membrane of outer hair cells was unique in that reaction product was never bound to this portion of the membrane. Subsequent to endocytosis, HRP was transported to the Golgi and its associated system of endoplasmic reticulum and lysosomes. The exogenous protein was sequestered in a striking accumulation of secondary lysosomes and multivesicular bodies which were restricted to the infracuticular region of the hair cells and persisted for periods of at least 72 h after introduction of HRP. It is not clear whether the hair cells were slowly degrading the HRP or if the lysosomal enzymes necessary for its breakdown were lacking. The pathway demonstrated by HRP uptake and intracellular transport may represent one route by which macromolecules requisite for basic metabolic or nutritional requirements of the hair cells are supplied from the perilymph.     

庆大霉素致豚鼠前庭毛细胞破坏后再生及功能恢复

为探讨哺乳动物前庭毛细胞破坏后能否再生及前庭功能能否恢复，以豚鼠为研究对象。实验组动物每日给予庆大霉素（１５０ｍｇ／ｋｇ）皮下注射，连续１０天，对照组动物给予等量生理盐水。于停药后的第２、４、１２和２４周分别处死动物，扫描电镜观察发现，停药２周椭圆囊和壶腹嵴毛细胞破坏最明显的区域可见到不成熟的毛细胞纤毛丛，至停药２４周后继续存在。与对照组相比，停药１２周毛细胞密度（毛细胞数／１００μｍ基底膜长度）下降明显（Ｐ＜０．０１），停药后２４周时毛细胞密度较１２周明显上升（Ｐ＜０．０５），但仍未达到正常水平（Ｐ＜０．０５）。另取豚鼠按上述方法进行庆大霉素注射，于注射前和停药后４、１２和２４周进行冰水诱发眼震电图检查，可见停药４～１２周平均慢相眼震速度（Ｖｍｅａｎ）明显下降（Ｐ＜０．０１），停药２４周后Ｖｍｅａｎ有明显恢复（Ｐ＜０．０５），但仍未达到正常水平（Ｐ＜０．０１）。提示：豚鼠周围前庭功能破坏后可以部分恢复，前庭毛细胞破坏后可以修复，其功能的恢复可能源于毛细胞再生。     

Differences in the distribution of F-actin in outer hair cells along the organ of Corti

There is evidence of differences in the structure, innervation and physiological responses between outer hair cells (OHCs) of the basal and apical turns of the mammalian cochlea. In this study we have used rhodamine-labelled phalloidin to investigate the differential distribution of F-actin in OHCs along the organ of Corti of the guinea pig. Isolated OHCs and surface preparations and cryosections of the organ of Corti were studied. F-actin was observed in stereocilia and the cuticular plate of all OHCs. In addition, some OHCs had a network of F-actin extending from the cuticular plate towards the nucleus. This infracuticular network was observed in most OHCs of the apical cochlear turns but was not seen in any OHCs of the basal turn. These microstructural differences between OHCs of the base and apex could be related to differences in OHC function between the apical and basal portions of the cochlea.     

新生小鼠耳蜗基底膜体外培养的实验研究

目的建立可靠的新生小鼠离体耳蜗基底膜的培养方法,为内耳的研究提供新的条件和模型。方法在显微镜下完整分离出新生1～5 d小鼠的耳蜗基底膜组织,采用组织贴壁法在无血清培养基中进行培养,免疫荧光法检测新生小鼠离体耳蜗基底膜培养组织中毛细胞及螺旋神经元的生长状态。结果新生小鼠耳蜗基底膜离体培养24 h后,显微镜下观察可见基底膜贴壁生长良好,外周有新生上皮细胞和成纤维细胞长出;高倍显微镜下可见耳蜗内外毛细胞和支持细胞等结构;免疫荧光法检测可见毛细胞和螺旋神经元生长良好,并能保持较长一段时间。结论组织贴壁法无血清培养新生小鼠离体耳蜗基底膜是一种理想的耳科实验造模方法。     

Changes in F-actin labeling in the outer hair cell and the Deiters cell in the chinchilla cochlea following noise exposure

It has been found that ‘conditioning' noise exposures can render the inner ear more resistant to traumatic noise exposures. To explore the possible mechanisms underlying this phenomenon, filamentous actin (F-actin), labeled by rhodamine-phalloidin, was examined in the chinchilla cochlea using confocal fluorescence microscopy. The conditioning noise was 0.5 kHz octave band noise (OBN) at 90 dB SPL for 6 h/day and the high-level noise was the same noise but at 105 dB SPL for 4 h. A variety of pathological changes were found in the chinchilla cochlea after exposure to noise. Subjects exposed to conditioning noise (1 day or 10 days) and only high-level noise showed an increase in F-actin labeling than unexposed controls. By contrast, subjects who had 5 days quiet after the 10-day conditioning exposure exhibited a decrease in F-actin labeling. Interestingly, subjects exposed to high-level noise with prior 10-day conditioning exposure also showed a decrease in F-actin labeling in the cuticular plate and the stereocilia. The F-actin decreases in the stereocilia and the cuticular plates may decrease the mechanical rigidity of the organ of Corti. A more pliable organ of Corti may have reduced the possibility of fracture or ripping of cell junctions during the motion of the basilar membrane induced by acoustic overstimulation.