【作者】 刘建平；

【导师】 王正敏； 戴春富； 张天宇；

【作者基本信息】 复旦大学 ， 耳鼻咽喉科学， 2005， 博士

【摘要】 第一部分:鼓室内注射庆大霉素在内耳的分布 一、鼓室内庆大霉素一次注射在内耳细胞的分布 目的:将庆大霉素同Texas Red连接形成gentamicin-conjugated Texas Red,即GTTR后,行豚鼠鼓室内注射GTTR,观察其在内耳的分布,比较一次鼓室内注射GTTR后,不同时间庆大霉素在前庭和耳蜗的分布情况。 方法:研究对象为豚鼠,行鼓室内GTTR注射一次,在注射后12h、24h、48h、3d、4d、7d、14d、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、血管纹庆大霉素分布情况,并进行荧光分布半定量分析。 结果:庆大霉素早期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浆中,支持细胞分布较少。在注射后第3天庆大霉素在内耳聚集达到最高峰,并在细胞内聚集较长时间,对临床治疗有指导意义。 结论:GTTR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。 二、不同方案鼓室内庆大霉素注射在内耳各细胞的分布的比较 目的:观察和比较豚鼠不同方案鼓室内注射GTTR后,庆大霉素在前庭和耳蜗的分布情况。 方法:A组鼓室内注射GTTR一次,14天后观察,B组鼓室内注射GTTR一次,28天后观察;C组鼓室内注射GTTR每天一次,连续四天,第一次注射后28天后观察;D组鼓室内注射GTTR每周一次,连续四周,第一次注射后28天后观察,Phalloidin染色后运用激光共聚焦扫描显微镜进行基底膜、椭圆囊、球囊、外半规管壶腹嵴扫描,观察GTTR荧光分布情况,并进行荧光半定量分析,比较各组荧光分布的差异。 结果:一次注射后,GTTR在注射后14天和28天在外毛细胞和、椭圆囊、球囊、外半规管壶腹嵴仍有较明显的聚集。每天一次,连续四天与每周一次,连续四周有非常明显的GTTR聚集。聚集主要在耳蜗的外毛细胞和前庭的感觉细胞。而在支持细胞未见明显累积。 结论:庆大霉素鼓室内注射后,在内耳聚集的时间较长,反复注射有累积效更多还原

【Abstract】 PART I The Distribution of Gentamicin in Inner Ear afterIntratympanic Gentamicin InjectionDistribution of gentamicin in Inner Ear after a single dose administration Purpose Uptake and accumulation of gentamicin by cells in the guinea pig inner ear after a single dose intratympanic application were investigated using a fluorescent probe --gentamicin-Texas-red conjunction (GTTR).Methods Adult guinea pigs (n=80) were administered a single dose of GTTR to the middle ear cavity through the intact membrane and survived for 12h,24h,48h,3d,4d,7d,14d and 28d. Laser confocal microscopy was used to observe the distribution of GTTR in the cochlear and vestibular cells after staining with phalloidin -alexa-488. TR and DMSO were injected into the tympanum as control.Results Soon after local drug administration, there was diffuse staining all tissue in the labyrinth. At later time the outer hair cells and sensory cells of vestibular organ were staining more densely than other cells in the inner ear. The peak level of fluorescent density was reached 3 days after local injection. The GTTR was seen in the infracuticular zone. This protocol produces no damage to the inner hair cells. Conclusion GTTR was a potential fluorescent probe to investigate the pharmacokinetics and mechanisms of gentamicin accumulation in local application over time. Distribution of gentamicin in inner ear after different protocol applications Purpose The uptake and accumulation of gentamicin in the guinea pig inner ear after different protocol treatments was compared using GTTR intratypanic injection. Methods Intratympanic injection of GTTR was applied to adult guinea pigs (n=32) using different protocol: in group A and B only one dose of GTTR was applied. In group C GTTR were injected once a day for four consecutive days and in Group D GTTR was given once a week for a total of four treatments. Laser confocal microscopy was used to observe the distribution of GTTR in the cochlear and vestibular cells after staining with phalloidin -alexa-488.Results After local drug administration using different protocols, the fluorescent was observed in the labyrinth especially in the outer hair cells and in the sensory cells of the vestibular. In Group C and Group D more GTTR accumulation was observed than that in Group A and B. But there was still density fluorescent 28 days after a singledose. The fluorescent of GTTR was also seen in the infracuticular zone.Conclusion Gentamicin accumulation was varied in local application using differentprotocols.PART II Damage of Inner Ear Cells after I ntratympanicGentamicin Injection using Different ProtocolsPurpose To compare the damage of inner ear cells after intratympanic gentamicininjection using different protocols.Methods Intratympanic injections of gentamicin were applied to adult guinea pigs(n=32) using different protocol administration: In Group A and B only one dose ofgentamicin was applied. In Group C gentamicin was injected one once a day for fourconsecutive days and in Group D gentamicin was given once a week for a total of fourinjection. Electrical scanning microscopy and Laser confocal microscopy were usedto observe the damage of gentamicin to the cochlear and vestibular cells.Results Damage can be seen in all of the four groups. In Group C and Group D thedamage were more severe than that in Group A and B. But there is less damage to theinner hair cells in Group A and B.Conclusion Damage was varied in local application using different protocols. Thedamage using a single lower dose to the inner hair cells was seen less severely thanother protocols in this study.PART III Uptake and accumulation of gentamicin By mouse hair cells in vitroPurpose To investigate the uptake and accumulation of gentamicin by mouse haircells in vitro.Methods The cochlear explants were prepared from the microdissected neonatalmouse cochlea. Cochlear explants were cultured with GTTR for different time. Laserconfocal microscopy was used to observe the distribution of GTTR in the cochlearand vestibular cells after labeling with phalloidin -alexa-488.Results Soon after culture, there was diffuse staining all tissue cells in the explants.At later time the outer hair cells were more staining than other cells in the explants.The peak level of fluorescent density was reached at 24 hours culture. The GTTR wasseen in the infracuticular zone of the hair cells.Conclusion GTTR and cochlea explants were useful methods to investigate thepharmacokinetics and mechanisms of gentamicin accumulation over time.更多还原