miR-431靶作用Eya4基因调控耳蜗功能的研究及双侧耳道闭锁患者Baha效果分析

【作者】 樊悦；

【导师】 陈晓巍； 朱大海；

【作者基本信息】 北京协和医学院 ， 耳鼻咽喉—头颈外科， 2014， 博士

【摘要】 第一部分基础研究miR-431靶作用Eya4基因调控耳蜗功能的研究目的研究miR-431对耳蜗功能的影响及与耳聋的关系,以补充阐明遗传性耳聋的病因。同时探讨miR-431调控耳蜗功能的可能分子机制,为遗传性耳聋的预防和治疗提供理论基础。方法主要采用Real-time PCR和microRNA原位杂交技术确定miR-431在小鼠耳蜗的时空表达谱。利用已构建的miR-431转基因(Transgenic, Tg)小鼠,进行听性脑干反应(ABR)测试评估小鼠听力,在动物水平了解miR-431对耳蜗功能的影响。利用小鼠基底膜铺片和耳蜗切片等技术探求miR-431转基因小鼠导致听力下降的耳蜗结构变化。应用生物信息学软件预测并利用双荧光素酶报告基因系统明确miR-431的靶基因。最后利用PC12细胞和原代培养的新生小鼠螺旋神经节细胞,在细胞水平进一步验证miR-431对神经元发育和分化的影响。结果miR-431在新生小鼠的耳蜗中的螺旋神经节、Corti器和血管纹中高表达,表达量随小鼠发育而逐渐减少。检测了A和B两个系13周龄的miR-431Tg小鼠和同窝的野生型(Wildtype, Wt)小鼠的听力,miR-431Tg小鼠听觉反应域明显高于Wt小鼠。miR-431Tg小鼠基底膜毛细胞可见点状缺失,螺旋神经节细胞分布较wt小鼠稀疏,石蜡切片上测量Rosenthal小管中螺旋神经节细胞密度显示,miR-431Tg小鼠明显低于wt小鼠。本研究发现,Eya4是miR-431的一个靶基因,miR-431Tg小鼠耳蜗中EYA4蛋白的含量减少。在PC12细胞中过表达miR-431抑制PC12细胞的分化。同时,miR-431Tg小鼠耳蜗原代螺旋神经节细胞成熟神经元数量和轴突长度降低。结论miR-431在小鼠耳蜗中过表达可导致小鼠听力下降。miR-431的高表达抑制了其靶基因Eya4的翻译,使EYA4蛋白形成受阻,进而导致螺旋神经节细胞数量减少,损害了听觉信号传导径路。第二部分临床研究双侧耳道闭锁患者Baha效果分析目的评估双侧先天性耳道闭锁患者应用骨锚式助听器(Bone-anchored hearing aid. Baha)后的听力效果及满意度,并分析术后并发症发生情况及预防和处理方法。方法对16例行单侧Baha植入术的双侧先天性耳道闭锁患者进行回顾性分析。分别测试患者在声场中裸耳与应用植入式Baha后的平均听阈,并应用普通话言语测听材料(Mandarin Speech Test Materials, MSTM)测试言语识别率(Speech Discrimination Scores, SDS)及言语识别阈(Speech Reception Threshold, SRT)。以Baha应用情况调查问卷和儿童格拉斯格受益列表(Glasgow children’s benefit inventory, GCBI)评估患者应用Baha后的生活质量提高程度和满意度。总结16例患者术后皮肤并发症的发生情况和处理方法。结果患者裸耳平均听阈为61.6±7.8dB HL,应用植入式Baha后平均听阈为23.8±5.9dB HL。在45dB HL和65dB HL给声强度下,裸耳SDS分别为6.7±7.4%和56.5±7.4%,应用植入式Baha后SDS提高至86.5±4.4%和90.1±3.4%。裸耳平均SRT为60.6±7.5dB HL,应用植入式Baha后降至24.7±5.0dB HL。Baha应用情况调查问卷显示患者应用Baha均获得满意效果,GCBI平均受益分数为45.6±14.4分。16例患者中共发生皮肤并发症5例。对1-2级皮肤反应者给予局部抗生素处理；3级反应者行二次手术切除增生皮肤及肉芽组织,其中1例患者更换9m桥基。结论双侧先天性耳道闭锁患者应用植入式Baha后听阈及言语识别能力得到明显提高,生活质量亦获得显著改善,患者主观满意度较高。桥基周围皮肤感染、皮肤过度增生是Baha植入术最常见的并发症。更多还原

【Abstract】 Part One Basic Research miR-431Involves in Regulating Cochlear Function by Targeting Eya4Objective To study the function of miR-431in cochleae in order to understand the relationship between microRNAs and hearing loss, and help clarify the cause of hereditary deafness. To provide the theoretical basis for prevention and treatment of hereditary deafness in the future.Methods We investigated the spatial-temporal expression profiles of miR-431in cochleae of mice using Real-time PCR and miRNA in situ hybridization. miR-431Transgenic(Tg) mice were established andauditorybrainstemresponses (ABRs) were detected to examine whether there was a difference of hearing ability between the Tg and Wt mice. We also used surface preparation technique of cochlear basilar membrane and cochlear sections to investigate the structural changes in the cochleae of Tg mice. We used bioinformatics softwares to predict the target mRNA of miR-431and confirmed the directinteractionbetweenmiR-431and the3’UTRof Eya4mRNA by luciferasereporterassay. Stable over-expression of miR-431wasachieved in PC12cells and the primary spiral ganglion neurons(SGN) of new born mice were cultured. Experiments in vitro further verified the inhibiting effects of miR-431in neurons.Results miR-431were highly expressed in SGN, Corti organ and striavascularis in cochleae of the new born mice and the expression were gradually reduced along with the developement. ABRs were detected in the Tg and Wt mice of A line and B line. The ABR thresholds were significantly higher in Tg mice compared to Wt mice. Few hair cells were missing in basal membrane of cochleae of Tg mice, and SGNs were sparse in Rosenthal canals of Tg mice. The density of SGNs of Tg mice was significantly lower than that of Wt mice. Eya4was confirmed as the target gene of miR-431by luciferasereporterassay and the EYA4content detected in cochleae of Tg mice was lower compared to Wt mice. The differentiation was inhibited in PC12cells in which miR-431were stably over-expressed. The proportion of mature neurons and the length of axons was also less in SGN primary culture of cochleae of miR-431Tg mice.Conclusion Over-expression of miR-431in cochleae of mice could result in hearing loss. Excessive miR-431inhibited the translation of its target mRNA-Eya4, leading to down regulation of EYA4protein. The deficiency of EYA4may be the reason of sparse SGNs in cochleae of Tg mice. Part Two Clinical Research The Efficacy of Bone-Anchored Hearing Adis in Patients With Congenital Bilateral Aural AtresiaObjective To evaluate the efficacy and satisfaction of bone-anchored hearing aids (Bahas) in patients with bilateral aural atresia. And to discuss the prevention and treatment of postoperative complications.Methods Baha was implanted during auricle reconstruction surgery, or after the auricle was rebuilt. Mean pure-tone threshold and speech audiometry test using Mandarin Speech Test Materials (MSTM) and results were compared among patients unaided and with Baha. Scores from the Baha user questionnaire and Glasgow Children’s Benefit Inventory (GCBI) were used to measure patient satisfaction and subjective health benefit. Retrospective analysis of16patients underwent Baha implantation were conducted to summarize the complications and treatment.Results The mean speech discrimination scores measured in a sound field with a presentation level of45dB HL were6.7±7.4%unaided and86.5±4.4%with Baha. Scores with a presentation level of65dB HL were56.5±7.4%unaided and90.1±3.4%with Baha. The unaided speech reception threshold was60.6±7.5dB HL and24.7±5.0dB HL with Baha. The mean pure-tone threshold of the patients was61.6±7.8dB HL unaided and23.8±5.9dB HL with Baha. The Baha application questionnaire demonstrated excellent patient satisfaction. The average benefit score of GCBI was45.6±14.4. Five patients suffered from skin complications. Patients had a Holgers Grade1-2skin reaction were cured by local application of antibiotics; and the patients experienced Holgers Grade3skin reaction were taken revision surgeries for excessive soft tissue growth. One patient replaced the Baha abutment with a longer9.0mm one.Conclusion The implanted Baha has been one of the most reliable methods of auditory rehabilitation for patients with bilateral aural atresia. It can improve the patient’s speech recognition performance and the quality of life. Infection and skin overgrowth around the abutment was the most common complications of Baha implantation.更多还原