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。     

经大鼠圆窗膜bFGF基因转导防治爆震性聋的实验研究

目的以阳离子脂质体介导bFGF(basic fibroblast growth factor,碱性成纤维细胞生长因子)基因,经完整圆窗膜途径转染爆震后的大鼠耳蜗,观察bFGF的表达及其对爆震性聋的防治作用。方法 SD大鼠30只,随机分为四组:空白对照组(n=6),仅接受155dB SPL脉冲噪声暴露20次;EGFP(enhanced green fluorescent protein,增强型绿色荧光蛋白)对照组(n=8),噪声暴露后即刻导入EGFP/阳离子脂质体复合物;bFGF治疗组(n=8),噪声暴露后即刻导入EGFP-bFGF/阳离子脂质体复合物;bFGF保护组(n=8),噪声暴露前3天导入EGFP-bFGF/阳离子脂质体复合物。各组动物分别于噪声暴露前及噪声暴露后1天、3天、7天、14天行ABR阈值测试。噪声暴露后14天耳蜗取材做基底膜铺片,荧光显微镜观察,并做bFGF免疫荧光染色验证bFGF的表达。结果爆震后1天,bFGF保护组ABR阈值低于空白对照组及EGFP对照组,且差异均有显著性意义(P<0.05)。爆震后3天、7天和14天,bFGF治疗组及bFGF保护组ABR阈值均低于两个对照组,且差异有显著性意义(P<0.05)。而爆震前及爆震后bFGF治疗组和bFGF保护组间的ABR阈值差异均无显著性意义(P>0.05)。术后14天,荧光显微镜直接观察及bFGF免疫荧光染色均检测到毛细胞内有bFGF的定位表达。结论将阳离子脂质体介导的bFGF基因经圆窗膜导入大鼠耳蜗能够表达,表达产生的bFGF对爆震所致的耳蜗损害有一定的保护作用,它能够减轻爆震后听。     

小鼠内耳圆窗基因导入的实验研究

目的研究携带增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）腺病毒（Ad-EG-FP）经由小鼠耳后圆窗径路导入内耳的可行性,分析EGFP在耳蜗内的表达特点。方法 19只健康的8～9周龄C57BL/6J雄性小鼠随机分为三组：Ad-EGFP组7只,人工外淋巴液组6只,两组通过耳后切口圆窗径路注射,分别导入Ad-EGFP和人工外淋巴液;空白对照组6只,未予处理。各组均于术前3日和术后7日行听性脑干反应（ABR）检查;术后7日取出耳蜗于荧光显微镜下观察EGFP在内耳的分布并行免疫组化观察EGFP在基底膜的表达。结果 3组动物术前ABR反应阈差异无统计学意义（P〉0.05）,术后7日,Ad-EGFP组ABR反应阈为62.86&#177;9.94dBSPL,人工外淋巴液组ABR反应阈为60.83&#177;9.70dBSPL,均较术前（37.86&#177;8.59和34.16&#177;8.04dBSPL）及空白对照组（40.83&#177;8.61dBSPL）高（P值均〈0.05）;空白对照组实验前后ABR反应阈无变化,Ad-EGFP组与人工外淋巴液组术后7天ABR反应阈差异无统计学意义（P值均〈0.05）。Ad-EGFP组Ad-EGFP导入后在基底膜上可见EGFP呈广泛表达,人工外淋巴液组和空白对照组基底膜未见荧光表达。结论外源性基因可经内耳圆窗导入并在耳蜗基底膜上广泛表达。     

耳后入路圆窗膜显微注射小鼠耳蜗基因转染新途径的研究

目的研究腺病毒携带目的基因经小鼠耳后人路圆窗膜显微注射途径耳蜗转导的可行性,为以小鼠作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12只C57BL／6J小鼠分为2组,实验组（8只）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）、对照组（4只）以人工外淋巴液经耳后入路圆窗膜显微注射注入耳蜗内。分别于术后5、14天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。结果术后动物存活10只（每组死亡1只）。实验组转染后耳蜗底回基底膜及螺旋神经节上目的基因有表达,14天组强于5天组。对照组耳蜗未见荧光表达。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。     

新型药物载体经圆窗膜途径给药的研究进展

内耳是一个复杂、结构精细、受严格调控的感觉器官,易受到内在和外在因素的干扰,导致暂时性或永久性的听力损伤[1].主要原因有噪声暴露、耳毒性药物、老龄化、自身免疫性疾病等[2-3],由其引起的听力减退、耳闷胀感、眩晕等症状极大地降低了患者的生活质量.目前临床上使用助听器、人工听觉植入等辅助设备来部分或完全恢复听力,但并没...     

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。     

荧光定量PCR测定水杨酸钠作用后大鼠耳蜗基因的表达

目的了解HSP27基因在大剂量水杨酸钠作用后大鼠耳蜗中表达水平,并探讨其在水杨酸钠耳毒性中的意义。方法将Wistar大鼠各15只分成水杨酸钠作用组和正常对照组,应用SYBR GREEN I实时监测的逆转录-聚合酶链反应(RT-PCR),检测大鼠耳蜗中HSP27基因在长期大剂量水杨酸钠作用后和正常对照组中的表达,并用管家基因β actin作为内参。结果水杨酸钠作用后大鼠耳蜗中HSP27基因的表达水平高于正常对照组,差异有统计学意义(P＜0.01)。结论SYBR GREEN I定量PCR法可以作为一种良好的方法对来源珍贵的微量组织的基因表达进行检测, 水杨酸钠能够明显诱导HSP27基因在大鼠耳蜗中的表达。     

Smad4 基因在野生型和基因缺陷小鼠耳蜗内的定位和分布

目的检测Smad4基因任成年小鼠耳蜗中是否有表达及表达部位的分布情况，以进一步研究Smad4基因在内耳发育以及听功能调控上的作用。方法动物由军事医学科学院发育和疾病遗传学研究室杨晓研究员提供．动物获取是在C57BL／6J和Blackswiss两种遗传背景的小鼠应用Cre—LoxP系统进行条件基因打靶，于小鼠胚胎早期在软骨细胞内将Smad4基因剔除，并生成同窝三种基因型小鼠-野生型Smad4＋/＋，杂合子Smad4＋／-，纯合子Smad4-／-用于本研究。对同窝三种不同基因型小鼠的耳蜗冰冻切片应用免疫组织化学方法染色观察．阴性对照为野生型小鼠耳蜗的冰冻切片并用PBS代替一抗．阳性对照为小鼠肾脏组织冰冻切片。结果Smad4在三种基因型小鼠耳蜗均有广泛表达，表达部位主要集中于血管纹、螺旋韧带、基底膜、盖膜、毛细胞、支持细胞、螺旋神经节细胞等处，其中血管纹和基底膜表达最为明显．Smad4在野生型和杂合型小鼠的耳蜗内表达情况基本一致，但纯合子小鼠耳蜗内表达与前两学相比为较低的水平．但未完全消失。结论Smad4在正常小鼠的耳蜗广泛存在，该基因是耳蜗中广泛表达的蛋白中的一种。作为Bmp4信号通路下游信号因子，它可能在骨性耳蜗的形成、内耳感觉细胞的分化成熟过程中起着重要的作用。     

胱硫醚-γ-裂解酶基因在大鼠耳蜗螺旋蜗轴动脉中的表达

突发性聋、噪声性聋以及老年性聋等都与耳蜗缺血有关。小脑前下动脉分出的螺旋蜗轴动脉是耳蜗供血的终末动脉，耳蜗的供血缺少侧支循环，当受到各种病理因素影响时，易发生微循环障碍而出现缺血性损伤。因此，研究耳蜗微循环调控特别是其自身调节能力是寻求解决缺血性耳蜗损伤的关键。     

三种病毒载体经蜗窗膜导入大鼠耳蜗的靶向分布比较

目的比较3种病毒载体经蜗窗膜注入大鼠耳蜗鼓阶介导报告基因在内耳的转染分布,甄选螺旋神经节细胞及其神经纤维基因靶向转移载体。方法分别将带有绿色荧光蛋白基因的慢病毒、5型-腺病毒、2型-腺相关病毒载体经大鼠耳蜗蜗窗膜缓慢注入鼓阶外淋巴,荧光显微镜观察病毒载体导入后3、7、14天绿色荧光蛋白基因在内耳表达部位及荧光强度。结果经蜗窗膜注入3种病毒载体能有效转染内耳多种类型细胞,相对其他两种病毒载体,5型-腺病毒载体高效转染螺旋神经节及其神经纤维,持续稳定表达2周以上。结论不同病毒载体经蜗窗膜导入后,在耳蜗内的表达分布存在明显差异,5型-腺病毒高效转染耳蜗神经,可作为未来听神经病变分子治疗的载体工具选用。     

Characterization of the Sheep Round Window Membrane

Intratympanic injection is a clinically used approach to locally deliver therapeutic molecules to the inner ear. Drug diffusion, at least in part, is presumed to occur through the round window membrane (RWM), one of the two openings to the inner ear. Previous studies in human temporal bones have identified a three-layered structure of the RWM with a thickness of 70–100 μm. This is considerably thicker than the RWM in rodents, which are mostly used to model RWM permeability and assess drug uptake. The sheep has been suggested as a large animal model for inner ear research given the similarities in structure and frequency range for hearing. Here, we report the structure of the sheep RWM. The RWM is anchored within the round window niche (average vertical diameter of 2.1 ± 0.3 mm and horizontal diameter of 2.3 ± 0.4 mm) and has a curvature that leans towards the scala tympani. The centre of the RWM is the thinnest (55–71 μm), with increasing thickness towards the edges (< 171 μm), where the RWM forms tight attachments to the surrounding bony niche. The layered RWM structure, including an outer epithelial layer, middle connective tissue and inner epithelial layer, was identified with cellular features such as wavy fibre bundles, melanocytes and blood vessels. An attached “meshwork structure” which extends over the cochlear aqueduct was seen, as in humans. The striking anatomical similarities between sheep and human RWM suggest that sheep may be evaluated as a more appropriate system to predict RWM permeability and drug delivery in humans than rodent models.     

Cochlear Aqueduct Flow Resistance Depends on Round Window Membrane Position in Guinea Pigs

The resistance for fluid flow of the cochlear aqueduct was measured in guinea pigs for different positions of the round window membrane. These different positions were obtained by applying different constant pressures to the middle ear cavity. Fluid flow through the aqueduct was induced by small pressure steps superimposed on these constant pressures. It was found that the resistance for fluid flow through the aqueduct depended on the round window position but not on flow direction. The results can be explained by special fibrous structures that connect the round window with the entrance of the aqueduct. It was also found that the equilibrium inner ear pressure depends on middle ear pressure, indicating that the aqueduct does not connect the inner ear with a cavity with constant pressure.     

Measurement of Basilar Membrane Motion During Round Window Stimulation in Guinea Pigs

Driving the cochlea in reverse via the round window membrane (RWM) is an alternative treatment option for the hearing rehabilitation of a nonfunctional or malformed middle ear. However, cochlear stimulation from the RWM side is not a normal sound transmission pathway. The basilar membrane (BM) motion elicited by mechanical stimulation of the RWM is unknown. In this study, the BM movement at the basal turn was investigated in both reverse via RWM drive and acoustic stimulation in the ear canal or forward drive in postmortem isolated temporal bone preparations of guinea pigs. During reverse drive, a magnet-coil was coupled on RWM, and the BM vibration at the basal turn and the movement of the incus tip were measured with laser Doppler vibrometry. During forward drive, the vibration of the incus tip induced by sound pressure in the ear canal resulted in BM vibration and the BM movement at the same location as that in the reverse stimulation was measured. The displacement ratio of the BM to RWM in reverse drive and the ratio of the BM to incus in forward drive were compared. The results demonstrated that the BM response measured in both situations was similar in nature between forward and reverse drives. This study provides new knowledge for an understanding of BM movement induced by reverse drive via the RWM stimulation.     

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.     

Round window membrane and labyrinthine pathological changes: an overview

The round window membrane is considered the most likely pathway from the middle to the inner ear. Various substances placed in the middle ear have been seen to pass through the round window membrane. Once toxic substances or inflammatory mediators such as cytokines and nitric oxide enter the inner ear, various inner ear sequelae such as labyrinthitis, endolymphatic hydrops, sensorineural hearing loss or more insidious diseases can occur.     

Gene Expression Profiles of the Rat Cochlea,Cochlear Nucleus,and Inferior Colliculus

High-throughput DNA microarray technology allows for the assessment of large numbers of genes and can reveal gene expression in a specific region, differential gene expression between regions, as well as changes in gene expression under changing experimental conditions or with a particular disease. The present study used a gene array to profile normal gene expression in the rat whole cochlea, two subregions of the cochlea (modiolar and sensorineural epithelium), and the cochlear nucleus and inferior colliculus of the auditory brainstem. The hippocampus was also assessed as a well-characterized reference tissue. Approximately 40% of the 588 genes on the array showed expression over background. When the criterion for a signal threshold was set conservatively at twice background, the number of genes above the signal threshold ranged from approximately 20% in the cochlea to 30% in the inferior colliculus. While much of the gene expression pattern was expected based on the literature, gene profiles also revealed expression of genes that had not been reported previously. Many genes were expressed in all regions while others were differentially expressed (defined as greater than a twofold difference in expression between regions). A greater number of differentially expressed genes were found when comparing peripheral (cochlear) and central nervous system regions than when comparing the central auditory regions and the hippocampus. Several families of insulin-like growth factor binding proteins, matrix metalloproteinases, and tissue inhibitor of metalloproteinases were among the genes expressed at much higher levels in the cochlea compared with the central nervous system regions.     

Round window gentamicin absorption: an in vivo human model

OBJECTIVE/HYPOTHESIS: Using a novel human labyrinthine sampling model, in vivo gentamicin absorption through the round window can be measured. STUDY DESIGN: A prospective study. METHODS: Gentamicin was delivered either transtympanically (preoperative) or through a facial recess approach (intraoperative). The lateral semicircular canal and vestibule were opened, and by means of a microsyringe, labyrinthine fluid was aspirated. A sample of serum was also drawn. In all patients cerebrospinal fluid was also drawn. The samples were analyzed using a standard chemistry analyzer. RESULTS: Intratympanic gentamicin diffused through the round window membrane and achieved concentrations in the labyrinthine fluid ranging from 0 to 16 mg/L. Intratympanic gentamicin was absorbed into the systemic circulation in 4 of 11 patients with serum levels ranging from 0.3 to 0.4 mg/L. No gentamicin was detected in the cerebrospinal fluid. CONCLUSIONS: Intratympanic gentamicin diffuses rapidly through the round window membrane and achieves significant levels in the inner ear. Thus, this new model can be used to assess round window permeability to clinically relevant medications such as steroids and ototopical antibiotics.     

Round window membrane visibility related to success of hearing preservation in cochlear implantation

Objectives: This study was designed to evaluate the relationship between the degree of round window membrane (RWM) exposure and hearing outcome.

Materials and methods: Forty-six ears with cochlear implantation (CI) were enrolled. The degree of RWM exposure was divided into Grade I (<25%), Grade II (25–50%), and Grade III (>50%). The hearing outcomes were evaluated at 1.5 and 12 months postoperatively.

Results: Twenty-seven ears were Grade I, 13 were Grade II, and 6 were Grade III. RW approach was used in all ears of Grades II and III and 20 ears of Grade I and cochleostomy was used in 7 ears of Grade I. The pattern of bony overhang was multidirectional in 41 ears. Threshold shift significantly decreased proportional to the increase of RWM exposure after CI. The mean RWM exposure was 32.1?±?24.4% in ears with more than partial preservation (n?=?17), and 13.3?±?11.7% in the other ears (n?=?6) at 12 months post-CI (p?=?.061). Age at CI differed significantly between ears that had more than partial preservation and the other ears at 1.5 months post-CI.

Conclusions and significance: Degree of RWM exposure and age at CI might be factors predicting hearing outcome after CI using the RW approach.