Math1基因内耳导入径路的探索研究

目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响,为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只,雌雄不限,体重250—300g。随机分成四组,完整圆窗膜组12只,鼓阶打孔组12只,各组分别设对照8只。实验组（24只）导入重组腺病毒携带的Math1基因及增强型绿色荧光蛋白基因（enhanced green fluorescent protein,EGFP）,对照组（16只）导入人工外淋巴液,所有动物均以左耳作为导入耳。术前及术后分别行听性脑干反应（ABR）检查。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片观察基因表达情况。结果完整圆窗膜组导入耳ABR阈值,术后5天各频率与术前比较无显著性差异（P〉0．05）;鼓阶打孔组导入耳ABR阈值,术后5天在2kHz、4kHz与术前比较无差异（P〉0．05）,8kHz较术前增高（P〈0．05）,16kHz、20kHz较术前明显增高（P〈0．01）,术后14天在16kHz、20kHz较术后5天时明显好转（P〈0．01）,但较术前仍有增高（P〈0．05）。转导成功率鼓阶打孔组为91．6％,优于完整圆窗膜组的50％。两种转导途径对目的基因在耳蜗内的表达部位和表达时间没有显著影响。结论完整圆窗膜途径及鼓阶打孔途径在转导成功率及听功能保护方面各有优劣。完整圆窗膜途径因其对耳蜗的损伤极小,在临床应用方面具有更好的发展前景。     

Math1基因内耳导入后噪声性聋豚鼠听功能改变观察

目的观察Math1基因内耳导入对噪声性聋豚鼠听功能的影响,探讨Math1基因过表达对噪声损伤耳蜗的生物学效应,为内耳基因治疗提供实验基础和理论依据。方法经脉冲噪声致聋的豚鼠45只（各频率ABR阈值均≥95dB SPL）,雌雄不限,实验开始时体草250～300g。随机分为3组：Ad—Math1-EGFP组（30只）;Ad—EGFP组（5只）;空白组（10只）。各组豚鼠在基因转导后4周、8周分别测试双耳ABR。测试完毕后处死动物,观察听泡及耳蜗尤炎性病变者记录听阈结果。结果Math1导入后4周,导入耳各频率ABR阈值低于对照耳（右耳）,也低于Ad—EGFP组及空内组,平均达到85dBSPL。Math1导入后8周,导入耳各频率ABR阂值低于对照耳（右耳）,也低于Ad—EGFP组及空白组,与4周时比较,进一步好转,平均达到75dB SPL。结论Math1基因内耳导入可使噪声导致全聋的豚鼠听功能部分恢复,为噪声性聋的治疗打开了新的思路和手段。     

The reaction of the guinea pig cochlea to perforations of the round window membrane with and without perilymph aspiration

Summary The influence of simple opening of the round window (RW) membrane and the effect of aspiration of perilymph on the electrophysiological characteristics of the cochlea was tested in guinea pigs by measurement of the compound action potentials. We found that perforations of the RW membrane failed to lead to either shortterm or long-term damage in cochlear function. There was only a slight spontaneous escape of perilymph but without measurable functional loss. Additional aspiration of perilymph led to entry of air into the basal turn and to an immediate loss of function of the cochlea. This regressed within 4 weeks in the middle- and low-frequency ranges. Measurable long-term damage persisted only in the high-frequency ranges. We attribute contradictory results of other authors to methodological errors which we avoided by a specific selection of healthy animals and the development of standardized operation, recording and measurement procedures.     

Ethanol infiltration into the stapedio-vestibular joint reduces low-frequency vibration of the ossicular chain and round window membrane in the guinea pig

Background: The synovial stapedio-vestibular joint (SVJ), which serves as a bridge between the stape and oval window, can be found in guinea pigs and most human adults. Unlike the fibrous SVJs in other animals, the contribution of the synovial SVJ to middle ear sound transmission remains unknown.

Aims/objectives: In this study, we investigate whether sclerosis of the synovial SVJ contributes to frequency-dependent vibration of the ossicular chain and round window membrane (RWM).

Materials and methods: A model of SVJ sclerosis model was established in the guinea pig using 75% ethanol. A laser Doppler vibrometer was then used to measure vibrations of the RWM and the long process of the incus (LPI) under pure tone sound stimulations of 0.25–16?kHz. The influence of SVJ sclerosis was analysed by comparing structural vibration displacement between the normal and sclerosis groups.

Results: Both LPI and RWM vibrations significantly decreased at low frequencies after infiltration of ethanol, which caused SVJ sclerosis.

Conclusions: SVJ sclerosis reduces low-frequency vibration of the ossicular chain and RWM in the guinea pig, which indicates that the synovial SVJ is vital to low-frequency sound transmission in the middle ear.

Significance: Providing useful data for further research regarding middle ear biomechanics.     

The effect of Hath1 r-expression in of guinea pig cochlea at one month after noise damage

Objective To study effects of Adenovirus-mediated Hath1 expression in guinea pig cochlea at one month after exposure to intensive noise. Methods Normal hearing guinea pigs, weighing 250-300g, received exposure to 200 rounds of impulse noise at 170 dB sound pressure level (SPL). The virus vector was inoculated into the left cochlea 1 month after noise exposure. Animals were tested using ABR and prepared for morphological examinations includeing immunocytochemistry and SEM 4 weeks after vector inoculation. Results The adenovirus mediated report gene expressed in the damaged area. There were no significant differences between treated and control animal in ABR threshold and morphologic changes. No new hair cells appeared in the Hath1 reated animals. Conclusion Forced hath1 ver-expression in the cochlea 1 month after noise exposure does not lead to appearance of new hair cells.     

Efferent effects elicited by electrical stimulation at the round window of the guinea pig

We report a technique for activating the efferent nerve fibres to the cochlea by electrical stimulation at the round window. Such electrical stimulation caused a reduction in the amplitude of the gross nerve response (N1) to a click presented after the electrical stimulus but did not alter the latency of the response. The reduction increased with increasing current strength above 200 microA and increasing rate of electrical pulses above 50 Hz. The effect was also dependent on the duration of the shock train and the pulse width. The reduction in N1 was most pronounced at low click intensities. Recovery of the N1 was almost complete about 80 ms after the end of the electrical stimulus. The effect of electrical stimulation in reducing the N1 amplitude could almost always be blocked by intraperitoneal injections of strychnine. Recovery from the strychnine block was observed when animals were maintained for periods of more than 60 min after the administration of strychnine. The ease of this technique allows it to be used to examine the effects of efferent stimulation on various aspects of cochlear function in the guinea pig.     

重组腺病毒构建及介导报告基因在豚鼠耳蜗中的表达

目的：带绿色荧光蛋白（GFP）基因的重组腺病毒,为利用重组腺病毒介导外源基因转导内耳提供依据。方法：通过细菌内同源重组方法构建携带有报告基因的重组腺病毒（Ad-GFP）,将重组腺病毒经耳蜗底回途径导入豚鼠耳蜗鼓阶外淋巴后,观察手术后不同时间GFP基因在豚鼠耳蜗内的表达、分布情况;检测手术前后听觉脑干诱发电位,观察手术和病毒对豚鼠听觉功能的影响。结果：构建的重组腺病毒经酶切电泳鉴定正确;豚鼠耳蜗底回途径导入腺病毒24 h后即有报告基因表达,3 d后最高,表达时间可持续2周以上;报告基因表达部位主要分布在血管纹、鼓阶外淋巴面的间皮细胞和耳蜗Corti器等部位;听觉脑干诱发电位（ABR）在各组动物手术前后无明显改变。结论：成功构建了携带GFP基因的重组腺病毒,通过该方法构建的腺病毒可以介导报告基因在豚鼠耳蜗中表达,并且对豚鼠听觉功能无明显影响,为内耳基因治疗提供了理论依据。     

腺病毒携带EGFP基因经完整圆窗膜转导豚鼠耳蜗的实验研究

目的研究腺病毒携带目的基因经完整圆窗膜途径耳蜗转导的可行性及安全性，为内耳基因治疗提供实验基础和理论依据。方法20只白色红目豚鼠，术前及术后分别行听性脑干反应（ABR）检查。实验组（12只）以重组腺病毒携带的增强型绿色荧光蛋白基因（enhancedgreenfluorescentprotein，EGFP），对照组（8只）以人工外淋巴液注入豚鼠圆窗龛内。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片，耳蜗冰冻切片观察。结果于圆窗龛内注入腺病毒携带目的基因的转导方法对听力无明显影响。转染耳蜗及对侧耳蜗内目的基因呈广泛表达。5天组表达产物最高，14天组逐渐降低。对照组耳蜗未见EPFP表达。结论于圆窗龛内注入腺病毒携带目的基因转导的方法对耳蜗无明显毒害作用，且能够将目的基因成功转导至双侧耳蜗组织并广泛表达。     

明胶海绵和透明质酸在地塞米松圆窗灌注中对豚鼠圆窗膜形态及功能的影响

目的圆窗分别放置明胶海绵与透明质酸,观察地塞米松圆窗灌注对豚鼠圆窗膜形态及功能影响。方法①36只豚鼠随机分为3组,Ⅰ组圆窗龛放置明胶海绵并在圆窗置管;Ⅱ组圆窗龛放置含2%透明质酸明胶海绵置管,Ⅲ组圆窗龛放置明胶海绵置管,Ⅱ组和Ⅲ组经圆窗置管向圆窗灌注地塞米松7d后。用光学显微镜和扫描电镜观察圆窗膜变化。36只豚鼠中随机取6只左耳作为正常对照（Ⅳ组）。②另取18只豚鼠,按给药时间分为1、4、7d3组,每组各取3只分为实验组和对照组,实验组圆窗放置透明质酸明胶海绵,对照组圆窗放置明胶海绵。各组经圆窗置管圆窗灌注地塞米松后,用LC-6A高效液相色谱仪测定外淋巴液中地塞米松浓度。结果光镜和扫描电镜检查发现Ⅰ、Ⅱ和Ⅲ组圆窗膜厚度和形态与正常对照Ⅳ组比较无明显差异（P〉0.05）;圆窗放置透明质酸明胶海绵后豚鼠外淋巴液中地塞米松含量明显高于明胶海绵组（P〈0.01）。结论①圆窗放置明胶海绵和透明质酸对豚鼠圆窗膜形态无明显影响。②透明质酸明胶海绵能提高地塞米松的圆窗膜通透性。     

Cochlear implantation via the round window membrane minimizes trauma to cochlear structures: A histologically controlled insertion study

Objective To evaluate cochlear implant trauma to intracochlear structures when inserting the electrode via the round window membrane.

Material and methods Eight fresh human temporal bones were evaluated histologically after insertion using two types of cochlear implant array. Bones underwent a special fixation and embedding procedure that allowed sectioning of undecalcified bone with the electrode in situ. Insertions depths were evaluated radiologically and histologically.

Results All arrays were found in the scala tympani of the cochlea. Basal trauma could be avoided in all but one specimen. The mean depth of insertion was 382.5°. Apically, only one implanted bone showed cochlear trauma exceeding lifting of the basilar membrane.

Conclusion Insertions through the round window membrane were shown to be atraumatic, even in basal cochlear regions. This route of insertion might be very effective for combined electric and acoustic stimulation of the auditory system.     

Concentration gradient along the scala tympani after local application of gentamicin to the round window membrane

OBJECTIVES: The distribution of gentamicin along the fluid spaces of the cochlea after local applications has never previously been demonstrated. Computer simulations have predicted that significant basal-apical concentration gradients might be expected, and histologic studies indicate that hair cell damage is greater at the base than at the apex after local gentamicin application. In the present study, gradients of gentamicin along the cochlea were measured. METHODS: A recently developed method of sampling perilymph from the cochlear apex of guinea pigs was used in which the samples represent fluid originating from different regions along the scala tympani. Gentamicin concentration was determined in sequential apical samples that were taken after up to 3 hours of local application to the round window niche. RESULTS: Substantial gradients of gentamicin along the length of the scala tympani were demonstrated and quantified, averaging more than 4,000 times greater concentration at the base compared with the apex at the time of sampling. Peak concentrations and gradients for gentamicin varied considerably between animals, likely resulting from variations in round window membrane permeability and rates of perilymph flow. CONCLUSIONS: The large gradients for gentamicin demonstrated here in guinea pigs account for how it is possible to suppress vestibular function in some patients with a local application of gentamicin without damaging auditory function. Variations in round window membrane permeability and in perilymph flow could account for why hearing losses are observed in some patients.     

阿霉素对豚鼠耳蜗外侧壁中多耐药基因1基因表达的影响

目的:探讨豚鼠耳蜗外侧壁中多耐药基因1(mdr1)是否对外源性的阿霉素(ADM)发生应激反应;ADM在耳蜗内淋巴液中积聚浓度的变化。方法:经静脉注射中毒剂量的ADM后,利用RTPCR技术半定量mdr1mRNA;采用高效液相色谱法(HLPC)检测内淋巴液中的ADM浓度。结果:①注射后24h时相点ADM组mdr1mRNA的量明显高于正常对照组,差异有统计学意义(P<0.05);而其4h时相点,与空白组相比差异无统计学意义;生理盐水组各时相点mdr1mRNA的量与空白组间差异无统计学意义;②内淋巴液中的ADM浓度4h时相点较24h时相点高,差异有统计学意义(P<0.05)。结论:mdr1是一种防御或应激基因,可被外源性ADM所诱导;其在耳蜗外侧壁中的表达,可能与减少耳毒性药物在内淋巴液中的积聚,增强耳蜗自我保护功能有关。     

The influence of Mossbauer source size and position on phase and amplitude measurements of the guinea pig basilar membrane

Phase and amplitude measurements were made from the incus and basilar membrane in guinea pig using the Mossbauer technique. The basilar membrane/incus ratio had a maximum of about 60 dB and a phase accumulation of between 9 and 12 radians to CF. Two source sizes were used (60 × 85 and 20 × 60 μ m) and the source was placed either on the modiolar edge of the basilar membrane or in the middle. Notches in plots of the basilar membrane/incus ratio occur at stimulus frequencies that appear to be associated with source size rather than position, suggesting that artefacts could be produced by the presence of the Mossbauer source.     

经完整圆窗膜途径EGFP基因在大鼠耳蜗中的表达

目的 研究经完整圆窗膜途径进行耳蜗基因转导的可行性及安全性，为内耳基因治疗提供实验基础和理论依据。方法 24只SD大鼠术前及术后分别行听性脑干反应(ABR)检查。实验组(18只)用阳离子脂质体携带的增强型绿色荧光蛋白(enhanced green fluoresent protein，EGFP)基因，对照组(6只)用生理盐水，注入置于圆窗龛处的明胶海绵内。分别于术后3、7、14天取双侧耳蜗标本做基底膜铺片观察。结果 于圆窗龛处放置明胶海绵的转导方法对听力无明显影响。转染耳蜗呈明显的绿色荧光。3天组表达产物最高，7天组逐渐降低，14天组更弱。对侧及对照组耳蜗均未见荧光表达。结论 于圆窗龛处放置明胶海绵进行基因转导的方法对听力没有影响，且能够成功转染耳蜗组织，是一种行之有效的方法。     

Permeability of round window membrane and its role for drug delivery: our own findings and literature review

We investigated the pharmacokinetics of caroverine in the perilymph, cerebrospinal fluid and plasma after systemic and local administrations in guinea pigs by using high-performance liquid chromatography. Auditory brainstem responses were measured to evaluate auditory functional effect. The results showed that local application was a both safe and efficient method. We further reviewed literature and pinpointed that the round window is ef-fectively local drug delivery means for future inner ear treatment.     

三种方法转染Math1基因效率和细胞毒性研究

目的比较腺病毒、脂质体、纳米材料三种不同类型的载体体外携带目的基因Math1对HEK293T细胞的转染效率及细胞毒性的大小,以期筛选理想的基因转染载体材料。方法选用重组腺病毒、LipofectamineTM2000、Superfect纳米材料作为转染载体,携带含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的Math1-EGFP基因及真核表达质粒pRK5-Math1-EGFP,并且根据不同转染载体说明步骤优化体外转染条件,分别转染293T细胞。在一定的时间内利用荧光显微镜观察细胞转染结果并计数阳性细胞,采用MTT(噻唑蓝)法检测三种不同载体体外细胞毒性,应用RT-PCR(逆转录聚合酶链反应)技术检测转染阳性细胞中目的基因Math1的mRNA表达情况。结果在优化的体外转染条件下,重组腺病毒载体介导的细胞转染率达到了94%以上,脂质体和商品化Superfect纳米材料转染的细胞中,荧光蛋白表达率分别为73%和80%以上,同时,商品化Superfect纳米材料在达到80%转染率的条件下,细胞存活率为90%。应用RT-PCR方法证实,三种载体转染细胞均有Math1基因的mRNA的表达。结论商品化Superfect纳米材料作为一种新型的、安全有效的纳米转染材料,能够成功携带耳聋治疗关键基因Math1转染293T细胞,以期在内耳基因治疗的研究当中得到有效的应用。     

缝隙连接阻断剂1-庚醇对豚鼠听觉的作用

目的：探讨缝隙连接在维持正常的听功能方面可能发挥的作用。方法：通过向豚鼠外淋巴中注入缝隙连接阻断剂1-庚醇（1-heptanol），然后检测给药前后豚鼠听性脑干诱发反应（ABR）阈值和潜伏期的改变，并借助透射电镜观察药物对豚鼠耳蜗中的缝隙连接结构的影响。结果：注射1-heptanol后，豚鼠ABR阈值显著提高，潜伏期延长（P〈0．01），并随时间推移呈动态升高趋势。同时，通过电镜可以观察到在豚鼠耳蜗中存在着大量的缝隙连接结构，其主要分布在毛细胞和其下的支持细胞间。1-heptanol可以使缝隙连接结构破坏，毛细胞水肿、变性。结论：缝隙连接阻断剂1-heptanol能明显影响豚鼠的听功能，缝隙连接结构在听觉形成过程中起着重要的作用。     

Ad5-atoh1/EGFP在体诱导近成年豚鼠耳蜗产生毛细胞样细胞

目的:在近成年豚鼠耳蜗内携带atoh1/EGFP基因的腺病毒是否能够将基膜上残留的支持细胞转分化为新的毛细胞。方法:选用体重在200～250g的健康花豚鼠12只,将构建同时表达atoh1和EGFP基因的E1/E3区缺失的人类免疫5型腺病毒5μl,通过耳蜗侧壁打孔灌注导入中阶内淋巴系统。其中6只于灌注2周后处死,6只灌注4周后处死,基膜铺片观察EGFP、毛细胞标记物myosinⅦa和Dapi核染色情况。结果:灌注2周处死的6只豚鼠中有2只在基膜外毛细胞外区域发现有散在的细胞核大、细胞体梭形的、表达EGFP的新生细胞。灌注4周的动物中有3只在基膜原外毛细胞位置和外毛细胞外区域发现有相似的同时表达EGFP和Myo-sinⅦa的新生细胞。结论:atoh1基因可以在近成年豚鼠体内将部分支持细胞转分化为毛细胞样细胞。这部分支持细胞位于外毛细胞位置和外毛细胞外区域的基膜上。     

腹侧听泡外入路小鼠耳蜗鼓阶基因导入的实验研究

目的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16只健康5周龄C57BL/6J小鼠,腺病毒组10只,以重组腺病毒携带有Hath-1和增强型绿色荧光蛋白基因（enhanced green fluorescent protein,EGFP）,人工外淋巴液组6只以人工外淋巴液,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。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对听力影响较小,且能够将目的基因成功转导至耳蜗组织并广泛表达。