豚鼠耳蜗的转基因表达及其意义

目的：探讨耳蜗转基因表达的方法及意义。方法：经圆窗将复制缺陷重组腺病毒（含β－半乳糖苷酶基因，Ad.LacZ）注入豚鼠一侧耳蜗（接种耳）鼓阶，1周后观察接种耳及对侧耳蜗（非接种耳）转基因表达及听力情况。结果：接种耳蜗及对侧耳蜗均有Ad.LacZ表达，其分布相似，但对侧耳蜗中Ad.LacZ基因表达相对较弱，双侧耳接种Ad.LacZ前后听力无明显改变。结论：对侧耳蜗的转基因表达可能对某些内耳疾病的转基因治疗具有重要意义。     

内耳转基因表达的实验研究

目的　了解内耳转基因表达的可行性。方法　将人复制缺陷重组腺病毒基因(Adenoviruses ,Ad ,含大肠杆菌 β 半乳糖苷酶基因LacZ基因 ,Ad5 LacZ)经豚鼠耳蜗蜗窗接种到鼓阶外淋巴后 ,观察在不同时间、不同内耳组织中LacZ基因的表达 (X Gal染色 )及Ad对豚鼠声反应 (听性脑干反应 )、听毛细胞 (扫描电镜 )的影响。结果　Ad介导的LacZ基因在内耳组织中的表达至少可持续 4周 ,其中在螺旋神经节细胞表达稳定 ,Corti器、前庭囊斑、壶腹嵴的毛细胞等也有较强的表达。Ad未对豚鼠声反应 (≤ 80 0 0Hz)造成明显的损伤 ,除耳蜗底回外 ,其余各回未见明显的毛细胞缺失。结论　腺病毒载体可成功地将LacZ基因转导致豚鼠内耳组织中 ,并且未对豚鼠声反应 (低、中频 )及听毛细胞造成明显损伤 ,这对未来的内耳基因治疗研究可能具有重要的参考价值。     

内耳转基因表达的实验研究

目的 了解内耳转基因表达的可行性。方法 将人复制缺陷重组腺病毒基因(Adenoviruses，Ad，含大肠杆菌β-半乳糖苷酶基因LacZ基因，Ad5．LacZ)经豚鼠耳蜗蜗窗接种到鼓阶外淋巴后，观察在不同时间、不同内耳组织中LacZ基因的表达(X-Gal染色)及Ad对豚鼠声反应(听性脑干反应)、听毛细胞(扫描电镜)的影响。结果 Ad介导的LacZ基因在内耳组织中的表达至少可持续4周，其中在螺旋神经节细胞表达稳定，Corti器、前庭囊斑、壶腹嵴的毛细胞等也有较强的表达。Ad未对豚鼠声反应(≤8000Hz)造成明显的损伤，除耳蜗底回外，其余各回未见明显的毛细胞缺失。结论 腺病毒载体可成功地将LacZ基因转导致豚鼠内耳组织中，并且未对豚鼠声反应(低、中频)及听毛细胞造成明显损伤，这对未来的内耳基因治疗研究可能具有重要的参考价值。     

腺病毒携带的LacZ基因在豚鼠耳蜗中的表达

目的 带有LacZ基因的腺病毒注入豚鼠耳蜗后,观察不同时间段LacZ基因的表达和分布情况及手术操作对听力的影响,为内耳基因治疗提供理论依据。方法 24只白色豚鼠术前及术后行听性脑干反应(ABR)检查。空白对照组经圆窗注入人工外淋巴液,实验组注入带有LacZ基因的腺病毒。分别于2天、l周、2周后取材。耳蜗标本经β-半乳糖苷酶(X-Gal)组织化学染色后做石蜡切片和耳蜗铺片。结果 腺病毒注入耳蜗后对听力影响不大。经X-Gal染色后整个耳蜗被染成蓝色。2天组表达产物最高,l周后逐渐降低。表达产物主要分布于柯蒂器的内外毛细胞、螺旋神经节细胞、基底膜下的间皮细胞。对照组均未着色。结论 通过圆窗注入腺病毒对听力没有影响,单一位点的接种就能使因子通过耳蜗液扩散到整个耳蜗。有效的基因转移在耳蜗是可行的,但表达相对短暂。     

耳蜗死区对感音神经性听力损失患者言语识别能力的影响

目的 探究耳蜗死区在感音神经性听力损失(sensorineural hearing loss,SNHL)患者中的存在情况及其对言语识别能力的影响.方法 采用纯音听阈测试筛选出41例(81耳)感音神经性听力损失患者,经均衡噪声阈值测试将患者分为有耳蜗死区组(35耳)和无耳蜗死区组(46耳),分别进行言语识别阈(SRT)和言语识别率(SDS)测试,分析81耳耳蜗死区的分布及其对言语识别能力的影响.结果 41例(81耳)感音神经性听力损失患耳中有35耳(43.21％,35/81)存在耳蜗死区,其中轻度SNHL患耳耳蜗死区检出率为0(0/11),中度SNHL患耳耳蜗死区检出率为24.1％(7/29),重度SNHL患耳耳蜗死区检出率为66.7％(24/36),极重度SNHL患耳耳蜗死区检出率为80.0％(4/5),不同听力损失程度耳耳蜗死区检出率差异有统计学意义(P＜0.05);高频耳蜗死区(16耳)明显多于低频耳蜗死区(8耳),但两者的言语识别能力差异无统计学意义(P＞0.05);有耳蜗死区患者的言语识别阈及言语识别率分别为61.63±16.76 dB HL,86.35％±12.03％,无耳蜗死区的患者分别为75.54±9.56 dBHL,64.97％±20.84％,二者间差异有统计学意义(P＜0.05).结论 听力损失越重,耳蜗死区检出率越高;高频耳蜗死区较低频常见,且存在耳蜗死区的感音神经性听力损失患者言语识别能力明显低于无耳蜗死区的患者.     

REZ-Ⅰ型人工耳蜗植入对成人残余听力的影响

目的 评估REZ-Ⅰ型国产人工耳蜗植入对成人残余听力的影响,探讨该人工耳蜗植入的听力学安全性及其损伤的特点.方法 回顾性分析复旦大学附属眼耳鼻喉科医院2009年9月至2009年11月间16例单侧REZ-Ⅰ型(22通道)人工耳蜗植入者在手术前后的纯音测听、听觉稳态反应(auditory steady-state response,ASSR)、畸变产物耳声发射(DPOAE)及听觉脑干反应(ABR)的资料,比较手术前后残余听力的变化情况.结果 纯音测听植入侧术后残余听力保留率为41.67%;术后植人侧250、500、1000、2000及4000 Hz的残余听力较术前下降,差异具有统计学意义,其中500 Hz下降较明显,平均下降15.3 dB HL,P<0.01;通过与非植入侧对比发现500和1000 Hz的听力损失有统计学意义(P值均<0.05).植入侧术后ASSR阈值在250和500 Hz处较术前升高,差异具有统计学意义(P值均<0.05)),经与非植入侧对比发现500 Hz处ASSR阈值升高有统计学意义(P值<0.05).DPOAE及ABR植入前后差异均无统计学意义(P值均>0.05).结论 REZ-Ⅰ型人工耳蜗植入会对植入侧残余听力造成一定程度的损伤.     

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。     

均衡噪声阈值检测法研究耳蜗死区在感音神经性聋患者耳蜗中的分布

目的探讨在感音神经性聋患者中是否存在耳蜗死区及其分布情况。方法采用均衡噪声阈值检测法检测82例(130耳)感音神经性聋患者和25例(50耳)正常听力者是否存在耳蜗死区,并比较耳蜗死区与性别、年龄、耳别、听力损失程度以及病程的关系。结果 82例感音神经性聋患者中有41.46%(34/82)存在耳蜗死区,正常听力者中无耳蜗死区,两者差异具有统计学意义(P<0.01)。耳蜗死区的存在在性别、年龄和耳别之间无明显差异,而不同听力损失程度、病程的感音神经性聋患者耳蜗死区的检出率差异有统计学意义(P<0.05),听力损失越重,病程越长,检出率越高。33.85%(44/130)受试耳存在耳蜗死区,其中高频区耳蜗存在死区者占23.08%(30/130),低频区耳蜗存在死区者占2.31%(3/130),高低频区同时存在耳蜗死区者占8.46%(11/130),高频耳蜗死区检出率远高于低频区。结论在感音神经性聋患者存在耳蜗死区中,耳蜗死区的存在与听力损失程度及病程有关,听力损失程度越重、听力受损时间越长,存在耳蜗死区的可能性越大。     

听神经病的病变部位探讨

目的探讨听神经病的发病部位.方法总结12例听神经病患者的病史、纯音及言语听力、镫骨肌反射、听性脑干反应(auditory brainstem response,ABR)、耳蜗电图、耳声发射及对侧声抑制试验的特点.结果12例的耳蜗电图-SP均消失.结论听神经病的发病部位在耳蜗的内毛细胞.     

耳蜗死区在老年性聋患者中的初步临床分析

目的探讨老年性聋患者中耳蜗死区及其分布情况。方法采用均衡噪声阈值检测法检测200例(370耳)老年性聋患者和25例(50耳)正常听力老年人中耳蜗死区分布情况,并比较耳蜗死区与老年性患者听力损失程度、病程以及合并高血压病之间的关系。结果 200例老年性聋患者中有38%(76/200)存在耳蜗死区。其中合并高血压病的老年性聋患者中有45%(56/124)存在耳蜗死区,是否合并高血压病组间耳蜗死区检出差异具有统计学意义(P<0.05)。正常听力老年人中无耳蜗死区,同老年性聋患者中比较差异具有统计学意义(P<0.01)。在不同听力损失程度、病程及合并高血压病的老年性聋患者中耳蜗死区的检出率差异有统计学意义(P<0.05)。结论在老年性聋患者中存在耳蜗死区,并且与听力损失严重程度、病程长短以及是否合并高血压病有关,听力损失程度越重、听力受损时间越长,存在耳蜗死区的可能性越大。     

Effects of trigeminal ganglion stimulation on the central auditory system

It is very important to determine if recombinant adenoviral vector (Ad) can damage the auditory hair cells in guinea pig cochlea after transgene expression. In this study, the scanning electron microscope was used to determine if there was loss of the auditory hair cells after Ad.LacZ (Ad5 containing Escherichia coli galactosidase) was inoculated into the cochlea through the round window membrane. Seven days later all inner and outer hair cells were found to express the LacZ gene. Except for the sparse loss of outer hair cells in the basal turn and the second turn, there was insignificant loss in the other turns. All inner hair cells were present. The damage to auditory hair cells resulting from intracochlear inoculation of Ad is limited, and this vector can be used as one of the ideal delivery tools in gene therapy of the cochlea.     

脑源性神经营养因子对耳蜗螺旋神经节的保护作用

目的　观察腺病毒携带的脑源性神经营养因子 (brainderivedneurotrophicfactor,BDNF)在豚鼠耳蜗中的表达 ,及噪声损伤后对螺旋神经节的保护作用。方法　 2 7只白色纯种豚鼠 ,暴露于135dBSPL ,4kHz的窄带噪声 4h。 7d后 ,12只经圆窗注入腺病毒携带BDNF(adenoviral mediatedBDNF ,ad BDNF) ,12只经圆窗注入ad LacZ ,3只注入人工外淋巴液。分别于 1、4、8周后取材 ,石蜡包埋中轴切片后 ,用免疫组化方法 (ABC法 )检测BDNF的表达。于光镜下计数螺旋神经节细胞。结果　在耳蜗各回中BDNF均有表达 ,4、8周组动物较 1周组动物表达弱。在 8周 ,注入ad BDNF组较ad LacZ组和人工外淋巴组螺旋神经节发生退行性病变的数目少 ,螺旋神经节细胞计数结果经统计学t检验 ,P <0 0 1,差异有显著性。结论　腺病毒携带的神经营养因子在耳蜗中能高效表达 ,在噪声损伤情况下腺病毒携带的脑源性神经营养因子对螺旋神经节有保护作用。该研究为基因治疗感音神经性聋提供了坚实的实验基础     

adenoviral-mediated hath1-egfp gene transfer into guinea pig cochlea through intact round window membrane

Objective To study expression of adenovira1-mediated Hathl-EGFP gene in the guinea pig cochlea after transfer through intact round window membrane (RWM), and to assess its effects on hearing. Methods Twenty adult guinea pigs were used, of which: 12 were surgically inoculated with AdHath1-EGFP in the bony groove of round window niche, and 8 with artificial perilymph. Auditory brainstem response(ABR) thresholds were determined in all animals before and 5 days after surgery. On post-surgery day 5 and day 14, animals were sacrificed and whole mounts of cochlea and fro zensections were examined. Results ABR tests showed no significant change of hearing after the surgery.Strong fluorescence staining in the cochleae was seen in Ad-Hathl-EGFP groups. The highest levels of gene expression were seen in the post-surgery day 5 group with tittle decrease on post-surgery day 14.The contralateral cochlea and those in the control groups were free of fluorescence staining. Conclusion The transgenic Hath1-EGFP can be effectively delivered into the inner ear through intact RWM, in an atraumatic manner.     

Protective effect of adenoviral-mediated brain derived neurotrophic factor gene on spiral ganglion]

OBJECTIVE: To observe the expression of brain derived neurotrophic factor (BDNF) in the cochlea of guinea pig, and assess the activity of BDNF in spiral ganglion cell following the damage of noise. METHODS: Twenty-seven guinea pigs were exposed to a 4 kHz narrow band noise at 135 dB SPL for 4 hours. At seven days after the noise exposure, twelve guinea pigs were inoculated with ad-BDNF, and twelve guinea pigs were inoculated with ad-LacZ, while three guinea pigs were inoculated with artificial perilymphatic fluid. The animals were sacrificed After 1 week, 4 weeks and 8 weeks, respectively. The cochlea was stained with immunohistochemisty (ABC method) to examine the expression of BDNF. The number of spiral ganglion cell was counted to assess the activity of BDNF. RESULTS: BDNF gene was expressed in whole cochlea, and the expression was obviously elevated 1 week after the adenovirus administration and reduced thereafter. The number of degenerated cells in ad-BDNF group in the eight weeks was lower than that in another two groups and the different between them was statistical significant (P < 0.01). CONCLUSION: Adenoviral-mediated brain derived neurotrophic factor can be expressed in a high level in the cochlea of guinea pig, and may prevent the cell of spiral ganglion from the damage of noise. This study lays the groundwork for alleviation of hearing loss using gene therapy.     

NT3重组腺病毒对耳蜗螺旋神经节的保护作用

目的　观察含有神经营养素 - 3(neurotrophin - 3,NT3)的重组腺病毒在豚鼠耳蜗中的表达及其在噪声损伤后对螺旋神经节的保护作用。方法　 2 7只白色纯种豚鼠 ,暴露于 135dBSPL、4kHz的窄带噪声中 4h。 7天后 ,12只经圆窗注入ad -NT3,12只经圆窗注入腺病毒携带的Lac2基因 (adenviruslacopron ,ad -LacZ) ,3只注入人工外淋巴液。分别于 1、4、8周后取材 ,石蜡包埋中轴切片后 ,用免疫组化方法 (ABC法 )检测NT3的表达。于光镜下计数螺旋神经节细胞。结果　在耳蜗各回中NT3均有表达 ,4、8周组动物较 1周组动物染色弱。注入ad -NT3组较ad -LacZ组和人工外淋巴组螺旋神经节发生退行性病变的数目少 ,螺旋神经节细胞计数结果经t检验有显著性差异 (P <0 .0 1)。结论　含有NT3的重组腺病毒在耳蜗中能高效表达 ,且在噪声损伤情况下对螺旋神经节有保护作用     

Differential gene expression profiles in salicylate ototoxicity of the mouse

CONCLUSION: This study demonstrated differential gene expression profiles in salicylate ototoxicity with oligonucleotide microarray. This study may also provide basic information on candidate genes associated with hearing loss and/or tinnitus or recovery after salicylate-induced cochlear dysfunction. OBJECTIVES: Salicylate ototoxicity is accompanied by temporary hearing loss and tinnitus. The purpose of the present study was to evaluate the gene expression profiles in the mouse cochlea with salicylate ototoxicity using DNA microarray. MATERIALS AND METHODS: The subject mice were injected intraperitoneally with 400 mg/kg of sodium salicylate; an approximate 30 dB threshold shift that was observed by auditory brainstem response was achieved 3 h after an injection of sodium salicylate and the hearing threshold returned to within normal range at 3 days. Differential gene expression profiles at 3 h after salicylate injection in comparison to the normal cochlea were analyzed with DNA microarray technology. RESULTS: No ultrastructural changes in the mice cochlea were observed by TEM at 3 h after salicylate injection. Microarray revealed that 87 genes were up-regulated twofold or more in the mouse cochlea with salicylate ototoxicity in comparison to the normal cochlea. Among these genes, increased expression levels of 30 functional genes were confirmed by semi-quantitative RT-PCR.     

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。     

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。