【作者】 徐金操；

【导师】 黄德亮； 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】 在现代社会中,由于先天性异常、病毒性疾病和耳毒性药物以及老年化退行性变等,导致前庭功能障碍患者不断增多。虽然前庭功能障碍有一系列潜在的不同因素,但前庭毛细胞的缺失是前庭功能障碍最主要的原因。目前,周围性前庭功能障碍的减轻主要依靠中枢性前庭功能的代偿,而不是前庭感觉上皮本身结构和功能的恢复。因此,如果我们能够找到一种方法来有效地替代损害或缺失的毛细胞,那么就有可能使周围性前庭功能障碍患者得到更有效的治疗。许多研究已经证实,成年哺乳动物耳蜗Corti器的毛细胞缺失后不能再生,而前庭感觉上皮具有一定的毛细胞再生能力。在一些动物模型中如豚鼠、大鼠以及灰鼠等,能够诱导前庭毛细胞的再生。Math1是哺乳动物内耳毛细胞发育过程中所必需的一个基因。已有研究证实,敲除Math1基因的小鼠,听觉和前庭毛细胞均未见发育,而在体外培养的Corti器中加入以质粒为载体的Math1基因后,产生了额外的毛细胞。用Math1在人类中的同源基因Hath1,同样重复出了上述结果。近来国外有研究报道,内耳导入以腺病毒为载体的Math1（Ad-Math1）基因后,药物性前庭功能障碍的小鼠出现了前庭毛细胞的再生和功能的恢复,使通过基因导入治疗前庭功能障碍成为可能。但是目前前庭毛细胞再生研究仍存在的问题是基因导入前庭最佳途径的选择,再生毛细胞的性质、功能和来源以及与突触的连接情况尚不清楚,这些问题仍阻碍前庭功能障碍基因治疗的临床应用。本文对前庭功能障碍动物模型的建立、基因导入前庭的方法和Math1基因对药物损害后大鼠前庭感觉上皮毛细胞再生的作用等进行了研究。本研究共分为3部分:一、新霉素对大鼠前庭毛细胞毒性作用的实验研究目的:研究新霉素对大鼠前庭毛细胞的毒性作用,为药物性前庭功能障碍动物模型的建立和相关研究提供参考。方法:将15只成年Wistar大鼠分为正常对照组、新霉素组和人工外淋巴液组,新霉素组大鼠在右耳通过耳蜗底转鼓阶打孔的方法在耳蜗内注入0.1%（g/100ml）新霉素5ul,人工外淋巴液组按同样方法注入人工外淋巴液5ul。正常对照组大鼠不做任何处理。处理3天后对动物进行颈髓硬膜外短声诱发电位（click-evoked potentials on thesurface of the cervical dura mater,CDM-CEP）检查和游泳试验,来评价前庭功能,然后将动物处死,进行组织形态学观察。结果:3天后新霉素组大鼠的前庭毛细胞即出现毁灭性破坏,并出现严重的前庭功能障碍。正常对照组大鼠的游泳时间为4.0±0.71s,新霉素组大鼠的游泳时间为10.2±1.64s,人工外淋巴液组大鼠的游泳时间为4.4±1.14s。在短声（Click）刺激下,正常大鼠引出CDM-CEP的阈值为85±3.54 dB（SPL）,阈值时的潜伏期为6.59±0.41s;新霉素组大鼠120dB（SPL）仍无法引出CDM-CEP;人工外淋巴液组大鼠引出CDM-CEP的阈值为90±5.0dB（SPL）,阀值时的潜伏期为6.68±0.31s。结论:新霉素对大鼠前庭毛细胞具有毁灭性的破坏作用,新霉素耳蜗内注射的方法可以建立理想的前庭功能障碍动物模型。二、基因导入大鼠前庭的方法及Ad-Math1内耳应用的安全性研究目的:探索前庭基因导入的方法和途径,并对Ad-Math1的安全性进行研究,为前庭功能障碍基因治疗的相关研究提供参考。方法:将25只成年Wistar大鼠分为正常对照组、缺失E1、E3基因且构建有Math1基因和绿色荧光蛋白报告基因的复制缺陷型腺病毒（adnovirus-Math1-enhanced greenfluorescence protein,Ad-Math1-EGFP）鼓阶导入组和前庭阶导入组。Ad-Math1-EGFP导入组大鼠在右耳通过耳蜗底转鼓阶或前庭阶打孔的方法导入物理滴度为2.1×10¹¹ v.p./ml的上述腺病毒（Ad-Math1-EGFP）5ul。正常对照组大鼠不做任何处理。在处理7天后对动物进行颈髓硬膜外短声诱发电位（click-evoked potentials on the surface of the cervical dura mater,CDM-CEP）和听性脑干反应（auditory brain-stem response,ABR）阈值检查和游泳试验,来评价前庭和耳蜗功能,并分别在处理后3天、7天将动物处死,进行组织学观察和形态学观察。结果:导入Ad-Math1-EGFP 3天后,Ad-Math1-EGFP前庭阶导入组的前庭终末器官及耳蜗均出现Ad-Math1-EGFP的转染;而Ad-Math1-EGFP鼓阶导入组的表达则局限于耳蜗,7天后仍未见前庭终末器官的表达;Ad-Math1-EGFP导入后,耳蜗及前庭器官的毛细胞及纤毛均未见破坏。导入7天后,正常对照大鼠的游泳时间为4.0±0.71s,Ad-Math1-EGFP鼓阶导入组大鼠的游泳时间为4.8±0.84s,前庭阶导入组大鼠的游泳时间为5.0±0.71s。在短声（Click）刺激下,正常对照组大鼠引出CDM-CEP的阈值为85±3.54 dB（SPL）,阈值时的潜伏期为6.59±0.41s,听性脑干反应（auditorybrain-stem response,ABR）阈值为37±4.47 dB（SPL）;Ad-Math1-EGFP鼓阶导入组大鼠引出CDM-CEP的阈值为91±5.48 dB（SPL）,阈值时的潜伏期为6.76±0.26s,ABR阈值为42±2.74 dB（SPL）;前庭阶导入组大鼠CDM-CEP的阈值为89±6.52 dB（SPL）,阈值时的潜伏期为6.78±0.26s,ABR阈值为40±3.54 dB（SPL）。结论:缺失E1、E3基因的复制缺陷型腺病毒对前庭和耳蜗毛细胞是比较安全的,可以作为基因导入比较理想的载体,耳蜗底转前庭阶打孔可以作为基因导入前庭的有效途径。三、Math1基因诱导成年大鼠前庭毛细胞再生的在体实验研究目的:探讨Math1基因对成年大鼠前庭毛细胞再生的作用及机制,为将来前庭功能障碍基因治疗的临床应用提供理论基础。方法:将35只成年wistar大鼠分为正常对照组、新霉素组和Math1基因导入组。新霉素组大鼠经鼓阶耳蜗内注射新霉素后,不导入Math1基因,而导入组动物经鼓阶耳蜗内注射新霉素两天后,通过前庭阶导入以缺失E1、E3基因的复制缺陷型腺病毒为载体物理滴度为2.1×10¹¹ v.p./ml的Math1基因（Ad-Math1）5ul,正常对照组大鼠不做任何处理。处理3月后,对三组动物进行颈髓硬膜外短声诱发电位（click-evokedpotentials on the surface of the cervical dura mater,CDM-CEP）检查和游泳试验,来评价前庭功能,并分别在处理后1、2、3月处死动物,将前庭囊斑取出进行组织形态学检查分析。结果:导入Ad-Math1基因一月后,导入组大鼠前庭未见毛细胞再生,而导入Math1基因两月后,损害的大鼠前庭感觉上皮可见毛细胞再生,导入三个月时的情况与两月时相似;新霉素组大鼠在以上各个时间点均未见前庭毛细胞再生。通过透射电镜进一步检查发现再生的前庭毛细胞绝大部分为Ⅰ型毛细胞,并与传入神经纤维之间有完整的突触连接,在新生的前庭感觉上皮中,可见有丝分裂现象。处理3个月后,正常对照组大鼠的游泳时间为4.0±0.71s,新霉素组大鼠的游泳时间为11.2±1.64s,Math1基因导入组大鼠的游泳时间为10.4±1.52s。在短声（Click）刺激下,正常对照大鼠引出CDM-CEP的阈值为85±3.54 dB（SPL）,阈值时的潜伏期为6.59±0.41s;新霉素组和Math1基因导入组大鼠在120dB（SPL）仍无法引出CDM-CEP。结论:Math1基因可以诱导大鼠前庭毛细胞的再生,再生的前庭毛细胞绝大部分为Ⅰ型毛细胞,并与传入神经之间有完整的突触连接,有丝分裂参与了前庭毛细胞再生的过程。结论1.新霉素对大鼠前庭毛细胞具有毁灭性的破坏作用,新霉素耳蜗内注射的方法可以建立理想的前庭功能障碍动物模型。2.缺失E1、E3基因的复制缺陷型腺病毒对前庭和耳蜗毛细胞是安全的,可以作为基因导入比较理想的载体,耳蜗底转前庭阶打孔可以作为基因导入前庭的有效途径。3.Math1基因可以诱导大鼠前庭毛细胞的再生,再生的前庭毛细胞绝大部分为Ⅰ型毛细胞,并与传入神经之间有完整的突触连接,有丝分裂参与了前庭毛细胞再生的过程。以上研究结果表明,以缺失E1、E3基因的复制缺陷型腺病毒为载体的Math1基因（Ad-Math1）可以诱导耳毒性药物严重破坏后大鼠前庭感觉上皮的毛细胞再生,提示前庭功能障碍的基因治疗是具有可行性的。更多还原

【Abstract】 Vestibular dysfunction patients increase gradually because of congenital abnormality,viral disorders,ototoxic drugs and aged degeneration in modern society.Although there are a diverse series of underlying causes for balance disorders,loss of vestibular hair cells represents a common cause of balance dysfunction.At presently,the peripheral vestibular dysfunction alleviation is depended on mostly central vestibular function compensation instead of vestibular sensory epithelium local restoration.So,the peripheral vestibular dysfunction patients maybe gain more effective therapy if we can find a method to replace the loss or damaged hair cells substantially.Many of researches previous demonstrated that mature mammalian hair cells of Corti organ were lack of capability of regeneration absolutely,but preserved a limit regeneration capacity still in vestibular sensory epithelium. Regeneration of vestibular hair cells can be induced in a variety of model systems including guinea pig and rat macular organs and the chinchilla crista ampullaris.One of the genes involved in the development of hair cells is the mammalian atonal homologue math1.Mice carrying a homozygous knockout of math1 failed to develop auditory and vestibular hair cells.Delivery of a plasmid vector expressing math1 to neonatal organ of Corti cultures produced supernumerary hair cells in vitro.These results were repeated using the human homologue of math1（hath1） and an adenovector（Ad） as the delivery vehicle. Most recently,hair cells were shown to be regenerated in adult mice vestibular neuroepithelium and restored vestibular function by math1 transfer in vivo after ototoxic damaged.So,it is possible that vestibular dysfunction will be alleviate by gene therapy.But some problems still exsist in the field of vestibular hair cell regeneration,for instances,property,resouce and function of new hair cells,and so on.These problems hamper vestibular dysfunction gene therapy in clinic presently.Our experiment carried out a series of researches,including preparation of vestibular dysfunction animal model and the approaches of gene transfer into vestibule and the effection of math1 in vestibular hair cells regeneration after aminoglycoside ototoxic insult.The present study can be divided into following three parts:Ⅰ.Research of ototoxicity of neomycin to rat vestibular hair cellObjective:To research neomycin ototoxicity for vestibule hair cell of rat, and provide the reference for the pharmacal vestibular dysfunction animal model developement.Methods:Fivteen mature wistar rats were divided into normal control group,neomycin group and artificial perilymph group（n=5）.Right ear of the neomycin group rat was administrated 5ul 0.1%（g/100ml）neomycin through the way of drilling scale tympanic of cochlear basal turn.The artificial perilymph group did the same way.As a control,the normal group do nothing to inner ear. To evaluate animal vestibular function,the click-evoked potentials on the surface of the cervical dura mater（CDM-CEP） and swimming time were recorded in all rats after treatment 3 days,and then histological and morphologic observation were carried out after animals sacrificed.Results:Vestibular hair cells were destroied and vestibular function was severely injuried in neomycin group after treatment 3 days.The swimming time was 4.0±0.71s in normal control group, 10.2±1.64s in neomycin group,and 4.4±1.14s in artificial perilymph group.The threshold and latency of CDM-CEP was 85±3.54 dB（SPL） and 6.59±0.41s in normal control group,90±5.0 dB（SPL） and 6.68±0.31 s in artificial perilymph group,but could not induce CDM-CEP at 120dB（SPL） level still in neomycin group.Conclusions:The neomycin possess the powerful capability of eleminating vestibular hair cells of adult rat;Neomycin intracochlear administration via scale tympani can develop an ideal vestibular dysfunction animal model.Ⅱ.Evaluation of the best way of Gene Transfer into Vestibule and safety of Ad-Math1 to inner earObjective:To explore the method that transfer gene into vestibule and observe the safety of Ad-Math1,in order to provide the base data for vestibular dysfunction gene therapy.Methods:Twenty-five mature wistar rats were divided into normal control group,the group of adenovirus（E1,E3-Deleted and carried math1 and enhanced green fluorescent protein report gene, Ad-Math1-EGFP） scale tympanic transfering group,Ad-Math1-EGFP scale vestibular transfer group.Right ears of the Ad-Math1-EGFP transfering group rats were deliveried 5ul Ad-Math1-EGFP(physical tite 2.1×10¹¹v.p./ml) into cochleas through the way of drilling scale tympanic or scale vestibular of cochlear basal turn.As a control,the normal group do nothing to inner ear.In order to estimate functional condition of vestibule and cochlea,the click-evoked potentials on the surface of the cervical dura mater（CDM-CEP） and threshold of auditory brain-stem response（ABR） and swimming time were recorded in all rats after treatment 7 days,animals were sacrificed after treatment 3,7 days respectively, and then histologic and morphologic observation were carried out after animals sacrificed.Results:All the vestibular end organs were transfected after treatment 3 days in the group of Ad-Math1-EGFP scale vestibular transfer,but could not observe the transfection in Ad-Math1-EGFP scale tympanic transfering group until 7 days after treatment,exception of cochlea.All animals’ morphologic observation showed that inner ear hair cells were normal after transfer;After treatment 7 days,the swimming tim ewas 4.0±0.71s in normal control group, 4.8±0.84s in Ad-Math1-EGFP scale tympanic transfer group,and 5.0±0.71s in scale vestibular delivery group.The threshold and latency of CDM-CEP was 85±3.54 dB（SPL） and 6.59±0.41s in normal control group,91±5.48 dB（SPL） and 6.76±0.26s in Ad-Math1-EGFP scale tympanic transfering group,and 89±6.52 dB（SPL） and 6.78±0.26s in Ad-Math1-EGFP scale vestibular delivery group;ABR（auditory brain-stem response） threshold was 37±4.47 dB（SPL）, 42±2.74 dB（SPL） and 40±3.54 dB（SPL） respectively in the groups above. Conclusions:The Ad（E1,E3-Deleted） is safe for vestibular and cochlea hair cells and can be used as an ideal vector of gene transfer.The cochleotomy of basal turn through scale vestibular can be served as effective way of transfering gene into vestibule.Ⅲ.Vestibular hair cell regeneration of adult rat induced by Math1 gene transfer in vivoObjective:To investigate effects and mechanisms of Math1 gene in vestibular hair cell regeneration of rats.Methods:Thirty five adult wistar rats wre divided into normal control group,neomycin group and Math1 gene transfer group.The rats of neomycin group did not transfer Math1 gene into inner ear followed neomycin intracochlear administration via scale tympani and the Math1 gene transfer group animals deliveried Math1 gene 5ul into vestibule through scale vestibuli after neomycin intracochlear injection via scale tympani two days and an adenovirus(E1,E3-Deleted and physical tite 2.1×10¹¹v.p./ml) as the delivery vector.As a control,the normal group do nothing to inner ear.In order to estimate functional condition of vestibule,the click-evoked potentials on the surface of the cervical dura mater（CDM-CEP） and swimming time were recorded in all rats after treatment 3 months.All animals were sacrificed respectively at three time points one,two and three months followed treatment, and then vestibular organs were dissected out and prepared for histological and morphologic examination.Results:Vestibular sensory epithelium did not occur hiar cell regeneration after Math1 gene transferred one month,but observe new hair cell in damaged vetibular sensory epithelium followed Math1 gene transfered two months and the similar phenomenon occured after Math1 gene transferred three months.The new hair cells were determined typeⅠnearly entire and formed intact synapsis with afference nerves by using transmission electron microscopy（TEM）.Furthermore,mitosis was detected in renewal vetibular sensory epithelium.After treatment three months,the swimming time was 4.0±0.71s in normal control group,11.2±1.64s in neomycin group,and 10.4±1.52s in math1 gene transfer group.The threshold and latency of CDM-CEP was 85±3.54 dB（SPL） and 6.59±0.41s in normal control group,but could not induce CDM-CEP at 120dB（SPL） level still in neomycin and math1 gene transfer group. Conclusions:Hair cell could regenerate induced by Math1 gene transfered in ototoxicity damaged vetibular sensory epithelium of adult rat.The new hair cells is identified as typeⅠnearly entire and formed intact synapsis with afference nerves.Mitosis is involved in vestibular hair cell regeneration procedure.Conclusions1.The neomycin possess the powerful capability of eliminating vestibular hair cell of rat;Neomycin intracochlear administration via scale tympani can develop as an ideal vestibular dysfunction animal model.2.The Ad（E1,E3-Deleted） is safe to vestibular and cochlea hair cell and can be used as an ideal vector for gene transfer.The cochleotomy of basal turn through scale vestibuli can be served as effective way of transfer gene into vestibule system.3.Hair cell could regenerate induced by Math1 gene transfered in ototoxicity damaged vetibular sensory epithelium of adult rat.The new hair cells were determined with typeⅠnearly entire and formed intact synapsis with afference nerves.Mitosis is involved in vestibular hair cell regeneration procedure.The results above demonstrated that vestibular hair cells of rats could regeneration by math1 gene transfer and an advirus（E1,E3-Deleted） as delivery vector,and show the possibility as gene therapy for vestibular dysfunction.更多还原