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

发布时间：2018-03-02 13:27

本文选题：miR-431　切入点：Eya4　出处：《北京协和医学院》2014年博士论文　论文类型：学位论文

【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。
[Abstract]:Part one basic study on the regulation of cochlear function by miR-431 targeting Eya4 gene
Objective to study the effect of miR-431 on cochlear function and its relationship with deafness, so as to further clarify the cause of hereditary deafness, and explore the possible molecular mechanism of miR-431 regulating cochlear function, so as to provide a theoretical basis for the prevention and treatment of hereditary hearing loss.
The method mainly uses Real-time PCR and microRNA in situ hybridization to determine the expression of miR-431 in mouse cochlea. The spectrum of space-time with the constructed miR-431 transgenic mice (Transgenic, Tg), auditory brainstem response (ABR) test and evaluation of mice hearing at the animal level to understand the impact of miR-431 on cochlear function. The cochlear structure changes by mouse basement membrane flatmount and cochlea slicing technology to explore miR-431 transgenic mice leads to hearing loss. Using bioinformatics software predicted target genes using dual luciferase reporter assay system. The use of miR-431 specific PC12 cells and primary cultured neonatal mouse spiral ganglion cells, to further verify the effect of miR-431 on neuronal development and differentiation at the cellular level.
The results of miR-431 in neonatal mouse cochlear spiral ganglion in the high expression of Corti and vascular pattern, the expression decreased gradually with the growth of wild type mice. The detection of A and B two 13 week old miR-431Tg mice and littermates (Wildtype, Wt) mice hearing, miR-431Tg mouse auditory response domain the basement membrane was significantly higher than that of Wt mice.MiR-431Tg mice hair cells punctate lack of spiral ganglion cells in WT mice distribution is sparse, paraffin section on the measurement of Rosenthal in tubular spiral ganglion cell density showed that miR-431Tg mice was significantly lower than that of WT mice. The study found that Eya4 is a target gene of miR-431, reduce the content of EYA4 protein miR-431Tg in mouse cochlea the over expression of miR-431 differentiation. Inhibition of PC12 cells in PC12 cells. At the same time, the number of primary miR-431Tg mouse cochlear spiral ganglion cells and axon length of mature neurons decreased.
Conclusion overexpression of miR-431 in mouse cochlea can lead to decreased hearing loss and high expression of.MiR-431, which inhibits the translation of target gene Eya4 and hinders the formation of EYA4 protein, which leads to a decrease in the number of spiral ganglion cells and a damage to the pathway of auditory signal transduction.
Analysis of the effect of the second part of clinical study on Baha in patients with bilateral aural atresia
Objective to evaluate the hearing effectiveness and satisfaction of patients with bilateral congenital atresia treated with Bone-anchored hearing aid. Baha, and analyze the occurrence and prevention and treatment of postoperative complications.
Methods 16 patients underwent unilateral Baha implantation of bilateral congenital aural atresia were analyzed retrospectively. The patients were tested in the sound field and the application of implantable ear bare average hearing threshold of Baha, and the application of Mandarin speech test materials (Mandarin Speech Test Materials, MSTM) test speech recognition rate (Speech Discrimination Scores, SDS) and the speech recognition threshold (Speech Reception, Threshold, SRT). With the application of Baha questionnaire and child benefit list (Glasgow children's benefit Glasgow inventory, GCBI) were assessed after the application of Baha to improve the quality of life and satisfaction. The incidence and treatment methods of 16 cases of patients with postoperative complications of skin.
Results the patients with naked ear average hearing threshold was 61.6 + 7.8dB HL, the average hearing threshold was 23.8 after application of implantable Baha + 5.9dB HL. 45dB and 65dB HL in HL to the sound intensity, naked ear SDS were 6.7 + 7.4% and 56.5 + 7.4%, application of implanted Baha SDS increased to 86.5 + 4.4% and 90.1 + 3.4%. average naked ear SRT 60.6 + 7.5dB HL, application of implanted Baha questionnaire to 24.7 + 5.0dB HL.Baha application showed that patients with Baha with satisfactory results, the average score was 45.6 + GCBI benefit from 14.4.16 patients had skin complications in 5 cases. Given the Bureau of the Ministry of antibiotic treatment of grade 1-2 skin reactions; two surgery hyperplasia of skin and granulation tissue in 3 stage reaction were performed, including 1 cases of patients with replacement of the 9m bridge.
Conclusion bilateral congenital aural atresia patients after application of implantable Baha hearing threshold and speech recognition ability was improved and the quality of life was significantly improved, patient satisfaction is high. The bridge surrounding skin infection, skin hyperplasia is the most common complication of Baha implantation.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.43

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