【作者】 徐延军；

【导师】 韩东一； 杨仕明；

【作者基本信息】 中国人民解放军军医进修学院 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 据世界卫生组织估计,全世界约有2.5亿人患有重度以上听力损失,其中2/3在发展中国家,我国是最大的发展中国家,听力障碍残疾人有2780万,60岁及以上为1540.5万,可以说老年性听力损失患者是我国最大的听力障碍群体。由于相当一部分老年性聋是由于毛细胞缺失引起,所以说毛细胞再生对于老年性聋的治疗有重要意义。随着分子生物学研究的发展,发现Math 1基因对于耳蜗发育过程中毛细胞的形成有决定性作用,进一步研究表明Math1有可能引起成熟哺乳动物毛细胞再生,但未见其能否在老年性聋动物模型中诱导毛细胞再生的报导。本研究拟行C57BL/6小鼠老年性聋动物模型评价;小鼠耳蜗外源基因导入途径及方法的研究;老年性聋C57BL/6小鼠内耳Math1基因导入后毛细胞样细胞的观察。第一部分C57BL/6小鼠内耳形态学和年龄相关性听力损失的研究[方法]为研究不同周龄C57BL/6小鼠内耳形态学及其ABR阈值变化。取C57BL/6小鼠3周、4周、12周、26周各15只,听性脑干反应(ABR)测试双侧2、4、8、16、20KHz ABR阈值测试。采用基底膜铺片后免疫组化染MyosinⅥ、Neurofilament,观察耳蜗毛细胞和神经丝的变化。扫描电镜观察耳蜗毛细胞及其静纤毛随年龄变化。冰冻切片HE染色观察corti’器毛细胞及支持细胞、血管纹、螺旋神经元细胞随年龄变化。[结果]随着年龄增长,C57BL/6小鼠各频率ABR阈值明显提高;顶转和底转内毛细胞缺失逐渐增多;神经丝染色渐淡;毛细胞静纤毛逐渐发生数量减少、增粗融合、倒伏等变化。到26周龄时已达到重度聋,各频率较3周组有显著统计学差异。顶转和底转内毛细胞有连续缺失,外毛细胞完全缺失,内毛细胞只有残存的少量静纤毛,粗细不均,倒伏明显。切片HE染色显示corti’器毛细胞及支持细胞、血管纹、螺旋神经元细胞随年龄变化也发生了明显的退行性变化。[结论]本研究对国产C57BL/6小鼠的内耳形态学进行观察,明确了其ABR阈值和内耳毛细胞的变化规律。说明当国产C57BL/6小鼠年龄达到26周时,ABR2、4、8、16、20KHzABR阈值已达到95dB(SPL)以上,外毛细胞已经完全缺失,可以用来作为毛细胞再生的模型,而不会受到残存内毛细胞的影响。血管纹、corti’器、螺旋神经元细胞随年龄变化也发生了明显的退行性变化,这给我们的实验提供了良好的老年性聋耳蜗微环境。也为国产C57BL/6小鼠老年性聋研究和活体毛细胞再生的动物模型提供了形态学理论依据,说明国产C57BL/6小鼠年龄达到26周时可以应用于老年性聋的毛细胞再生研究。第二部分C57BL/6小鼠外源基因内耳导入路径与方式及内耳转染的研究一C57BL/6小鼠耳后入路各种导入方式为了研究腺病毒携带目的基因经小鼠耳后入路各种导入方法的可行性,为以小鼠为动物模型的内耳基因治疗提供实验基础和解剖学依据。[方法]我们采用经圆窗膜显微注射腺病毒携带目的基因导入、刺破圆窗膜-明胶海绵介导载体导入、用浸泡治疗介质明胶粘贴圆窗膜的方法,将携带的增强型绿色荧光蛋白基因的重组腺病毒或人工外淋巴液导入C57BL/6J小鼠耳蜗。耳后入路磨开听泡后上骨壁,暴露圆窗龛,经圆窗膜显微注射注入耳蜗内。分别于术后7天ABR测听后取双侧耳蜗标本做基底膜铺片,在激光共聚焦显微镜下观察GFP表达,同时用扫描电镜、MyosinⅥ染色观察导入后耳蜗形态学变化。[结果]术后动物存活较好,经圆窗膜显微注射导入后听力丧失,其余各组听力保存较好。各组腺病毒导入后,转染耳蜗底转基底膜及螺旋神经节上目的基因有表达。对照组耳蜗未见荧光表达。但是用浸泡治疗介质明胶粘贴圆窗膜方法导入后,外源基因表达过于局限。圆窗膜显微注射导入和刺破圆窗膜-明胶海绵介导载体导入后外源基因表达强烈,但是刺破圆窗膜-明胶海绵介导载体导入后外毛细胞受损严重。[结论]耳后入路操作简单、损伤小、易于暴露圆窗龛。耳后入路圆窗膜显微注射腺病毒携带目的基因转导的方法能够将目的基因成功转导至耳蜗组织并表达,同时可以满足单纯的形态学研究。二腹侧听泡外入路小鼠耳蜗鼓阶基因导入为研究腺病毒携带目的基因经腹侧听泡外入路转导耳蜗鼓阶底转的可行性及目的基因的表达特点,为内耳基因治疗提供实验基础和理论依据。[方法]将3周龄C57BL/6小鼠,以重组腺病毒携带的增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)或人工外淋巴液经腹侧听泡外入路导入耳蜗鼓阶底转。分别于术后第7天分别行听性脑干反应(ABR)检查后取双侧耳蜗标本做基底膜铺片、耳蜗冰冻切片观察基因的表达。同时用扫描电镜和铺片MyosinⅥ染色以观察毛细胞纤毛和胞体受损情况。[结果]经腹侧听泡外入路转导耳蜗鼓阶底转的转导方法对听力及毛细胞影响较小。腺病毒组耳蜗内目的基因呈广泛表达。对照组耳蜗未见荧光表达。[结论]经腹侧听泡外入路转导耳蜗鼓阶底转的转导方法可以有效保护听力及毛细胞,且能够将目的基因成功转导至耳蜗组织并广泛表达。第三部分老年性聋模型小鼠C57BL/6耳蜗鼓阶外源基因Math1导入后出现毛细胞样细胞现象的观察为了观察老年性聋C57BL/6小鼠(8月龄)耳蜗鼓阶外源基因Math1导入后能否在老年性聋的耳蜗微环境诱导出新生的毛细胞样细胞,为Math1基因治疗老年性聋进行探索性研究。[方法]将8月龄c57BL/6小鼠,以携带Math1基因的重组腺病毒(实验组)或只携带GFP基因的重组腺病毒(对照组)经腹侧听泡外入路导入耳蜗鼓阶底转。分别于术后第3.5个月分别行听性脑干反应(ABR)检查后取双侧耳蜗标本做基底膜铺片和耳蜗冰冻切片MyosinⅥ染色观察有无新生毛细胞样细胞出现,部分耳蜗作扫描电镜观察。[结果]实验组和对照组最大刺激均无ABR波形引出,扫描电镜也未能观察到纤毛结构。但是在Math1基因导入后,外毛细胞区域有MyosinⅥ染色阳性的新生毛细胞样细胞,部分动物Math1基因导入后耳蜗内有核分裂现象存在,发生核分裂的细胞团MyosinⅥ染色阳性。表明外源Math1基因具有在老年性聋的耳蜗微环境诱导出新生的毛细胞样细胞的能力,但是尚不能使听功能有效恢复。[结论]Math1基因导入在基因治疗老年性聋方面有潜在应用价值,但是还需与其他治疗性药物或基因联合应用。更多还原

【Abstract】 Presbycusis influences human being severely.Hair cell degeneration still on clinical impossible recovery present.So regenaration of hair cells is the key point of treatment of presbycusis.In order to test the probability of Math1 gene induces hair cell regeneration in cochlea of presbycusis mice,we prosecuted the study and divided into three parts as following.Part one:Age-related Hearing Loss and Morphological Study of Inner Ear in C57BL/6 Mice Inbred Mouse StrainsTo assess the dynamic age-related hearing loss of inbred mice C57BL/6 and investigate the morphological changes in the inner ear of varied age.In the present study,we tested ABR threshold to evaluate at 2、4、8、16、20kHz in C57BL/6 of 3、4、12、26 weeks old respectively.Morphological changes of inner ear hair cells were qualitatively determined by use of scanning electron microscopy and histochemistry staining of MyosinⅥwith Neurofilament.The cochlea were sectioned at 6um and then stained with HE.ABR threshold of C57BL/6 gradually increased with age increasing.Contrasted that with 3 weeks old mice,ABR threshold of 26 weeks old mice were dramatically inecreased at 2、4、8、16、20kHz.In scanning electron microscopy and histochemistry staining,the hair cells at top turn and basal turn of cochlea missed and the stereocilia gradually became lossed,fussed,disorganised with age increasing.In 26 weeks old,the outer hair cells almost all missed.In HE section,both the SV and the SG were showed atendency for progressive atrophy to develop with age.Our study shown there is significant age-related hearing loss and hair cell missing in C57BL/6 inbred mouse strain.C57BL/6 inbred mouse strain could be served as animal models of age-related hearing loss for further study of hair cell regeneration. Part tow:The Study on Path Ways of Inner Ear Gene Transfection in MiceSection one:Adenoviral Vectors Mediate Inner Ear Gene Transfection by Postauricular Approach with Three Methods in C57BL/6 MiceTo assess the feasibility of adenoviral vectors mediate cochlear gene tranfer by postauricular microinjection through the round window and application of gelatin soaked with therapeutic agent to the round window membrane with or without puncture of the round window membrane in mouse.Three weeks old C57BL/6 mouse were used for this study.Group 1 mice were implanted with Ad-EGFP or artificial perilymphatic fluid by postauricular microinjection through the round window membrane.Group 2 mice were implanted by postauricular application of gelatin soaked with therapeutic agent to the round window.Group 3 mice were implanted by postauricular application of gelatin soaked with therapeutic agent to the round window with puncture of the round window membrane.On postoperative day 7,the animals were sacrificed after ABR test.The surface preparation was observed,and then stained with MyosinⅥ.Other cochlea specimens were observed with SEM.Bright green fluorescence in the cochleae was observed in Ad-EGFP groups.Gene expression in groupl and group3 was higher than that in group 2.However,control groups were none any of fluorescence.The postauricular approach of mouse cochlear gene tranfer is simple for operation and with little side-effect.The technique of transgenic delivery into the inner ear through RWM by microinjection is feasible and effective,but damage to hearing transduction.Section two:Intact Tympanic Buila Cochlear Gene Delivery into the Scala Tympani of MouseTo assess the feasibility of adenoviral vectors mediate cochlear gene tranfer by ventral approach microinjection into the scala tympani of mouse with intact tympanic bulla and the expression of EGFP.Three weeks old C57BL/6 mice were used for the study and were microinjected with Ad-EGFP or artificial perilymphatic fluid in the scala tympani.Auditory brainstem response(ABR) thresholds were determined in all animals 7 days after surgery.Then the animals were sacrificed and the surface preparation and the section of cochleae were observed.The surface preparation was observed,and then stained with MyosinⅥ.Other cochlea were observed with SEM.ABR measurement showed that the operation had no significant injury on the hearing.Bright green fluorescence in the cochleae was observed in Ad-EGFP groups.Artificial perilymphatic fluid and control groups were free of fluorescence.The technique of transgenic delivery into the inner ear by ventral approach microinjection into the scala tympani with intact tympanic bulla is feasible,atraumatic and effective.Part three:Cochlear on Hair Cell Like Cells Regeneration in Presbycusis C57BL/6 Mice after Math1 Gene Inner Ear TransfectionIn order to test the probability of Mathl gene induces hair cell like cells regeneration in cochlea of presbycusis C57BL/6 mice,8 mounths old C57BL/6 mice were used for this study and were microinjected with Ad-MathlorAd-EGFP in the scala tympani by ventral approach.Auditory brainstem response(ABR) thresholds was determined in all animals 3.5 mounths after surgery.Then the animals were sacrificed and the surface preparation and the section of cochleae stained by MyosinⅥwere observed.Other cochlea were observed with SEM. Positive cells of MyosinⅥin the cochleae of Ad-Math1 groups were found.Some positive cells of MyosinⅥwere proliferate and their karyons were dividing.This results show Math1 gene can induce regeneration of new hair cell like cells in cochlea of presbycusis C57BL/6 mice.But the hearing of the mice with Math1 transfection were not improved.So Math1 gene with other factors may be necessary to improve hearing.更多还原