【作者】 高儒真；

【导师】 陈晓巍； 刘东；

【作者基本信息】 北京协和医学院 ， 耳鼻咽喉—头颈外科， 2012， 博士

【摘要】 目的：世界范围内约有2.78亿人患有中度以上的听力损失,在中国听力语言残疾患者达2780万人。聋病防治主要包括传导性耳聋和感音神经性耳聋两个方面。半个世纪以来,传导性耳聋已经取得成熟的防治经验,感音神经性耳聋的预防和治疗是聋病基础研究的热点与难点。临床上,很多感音神经性耳聋为内耳发育异常所引起。内耳畸形和耳聋病程复杂,功能为不可逆缺失,治疗方法相对局限。寻找更多的内耳畸形、耳聋致病基因,探索致畸致聋机制,并筛选针对发病机制特异性治疗制剂,已成为耳科学基础与临床研究者的共同责任。LM04基因编码一种转录调节因子,小鼠耳泡中组织特异性敲除LM04,可见半规管发育异常、半规管嵴发育不良。因此LM04得到更多学者的关注。斑马鱼内耳发生机制以及前庭形态、功能保守,胚胎透明、易于操作,是研究内耳发育畸形和耳聋的优势模型。本实验主要研究lmo4基因对斑马鱼内耳发育的影响,并探索影响机制,为内耳畸形、感应神经性耳聋的防治奠定理论基础。方法：本文主要采用斑马鱼胚胎内注射寡核苷酸Morpholino (MO)和mRNA的方法构建lmo4基因低表达和过表达模型；通过表型观察、原位杂交、免疫组化等方式研究lmo4基因对斑马鱼内耳发育的重要作用；通过筛选与内耳结构发生密切相关的基因表达变化研究lmo4基因影响内耳发育的机制；并针对机制筛选可治疗挽救表型的制剂。结果：斑马鱼中lmo4的两个同源基因分别是lmo4a和lmo4b,两者均为母源性基因。10hpf,lmo4a主要在中胚叶及神经板中表达；14hpf开始表达于耳基板、耳泡及周围基质；随着耳泡发育,lmo4a表达范围与毛细胞区域部分重叠,并标记与半规管发生紧密相关的3个半规管嵴及半规管突起。lmo4b在耳泡、周围基质、及侧线中持续表达。通过Morpholino低表达lmo4b后未观察到内耳表型,我们后期的研究主要集中于lmo4a在内耳的功能。通过Morpholino低表达lmo4a后发现前基板发育受阻；耳泡大小、毛细胞数目、听平衡神经节内细胞数目呈等比减少；同时伴有特异的半规管发育异常(仅水平半规管突起形成)；与半规管发育紧密相关的bmp4,bmp2b明显上调,同时下调BMP信号可很大程度挽救低表达lmo4a诱导的半规管不发育表型。结论：低表达lmo4a诱导的耳泡大小、内耳毛细胞和神经节的数量减小可归因于lmo4a对前基板早期发育的影响；而半规管畸形是由于BMP信号上调导致；耳泡内注射DM (AMP-activated protein kinase inhibitor, Dorsomorphin)小分子可下调BMP信号,促进半规管发育,挽救畸形。本实验探讨了lmo4a基因在斑马鱼内耳发育的重要作用和作用机制,耳泡内注射小分子DM可促进半规管发育,挽救半规管畸形。更多还原

【Abstract】 Deafness effects the health of human seriously. Hearing loss affects approximately27million in the china alone and has a high incidence-about278million people suffer from moderate to severe hearing loss in the world. It can be safely assumed that all children with congenital sensory hearing loss would have detectable abnormalities in their inner ears if they could be examined histologically. Development of therapies to treat hearing loss and balance disorders iscomplicated by the diversity of disease processes leading to functional loss and the current approaches available for treatment. Despite the significant impact of hearing and balance disorders on the general population there are currently no dedicated pharmaceuticals that target the inner ear.Incidence reduction, early detection, and early treatment of hearing loss have become a common interest of society. Prevention and treatment of Inner Ear Malformations and sensorineural deafness basic research is still hot and difficult.The role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. In mouse, LMO4is initially expressed in the dorsolateral portion of the otic vesicle and its expression persists in the semicircular canals, macula, crista throughout embryogenesis indicating its important role in the mouse inner ear development. Targeted disruption of Lmo4resulted in the dysmorphogenesis of the vestibule and in the absence of three semicircular canals, anterior and posterior cristae.Zebrafish has two homologous genes Imo4a and Imo4b. Transcripts of both genes were present in unfertilized eggs, suggesting a maternal origin. Imo4a expression is rather dynamic, mainly in the developing mesoderm and neural plate at late gastrula and segmentation stages. The onset of Imo4a in the otic organs was detected weakly in the otic placode at14hpf. From then on, Imo4a is expressed outside the developing otic vesicle, as well as at anterior-dorsal tip and the ventral part of the vesicle. At24hpf, Imo4a expression likely includes some ventral cells of the vesicle and perhaps newly emerged hair cell precursors. At36hpf, Imo4a expression is seen in the anterior, ventra-lateral and posterior positions, perhaps marking the3developing cristae. The epithelial protrusions, which form the future semicircular canal system, start to undergo morphological change at48hpf, and weak Imo4a expression is detected in the apical protrusion.No obvious otic phenotype was detected in Imo4b morphants,so further research was focus on the function of Imo4a in the inner ear.Loss of Imo4a function leads to proportional reduction of otocyst size, hair cells statoacoustic ganglia. The markers that can highlight the preplacodal ectoderm (PPE. eyal and six4.1), or early sensory cells that derived from the PPE(i.e., ngnl, atoh1b and dlx3/4b) are down-regulated suggesting there is an overall decrease of gene expressions in the caudal PPE and PPE derived cells in the morphants and there is an early otic specification defect that leads to a general defect of inner ear functions.However, a failure to grow the semicircular canals (SSCs) in Imo4a morphants suggests us a direct link between Imo4a and SSC epithelial budding event.Because bmp4, bmp2b,which, are normally expressed in sequence at the epithelial protrusion buds of the otic vesicle, are all elevated in lmo4aMO ears, we reason that up-regulation of Bmp signaling may prevent the outgrowth of the protrusions in the morphants. As our presumption,injection AMP-activated protein kinase (AMPK) inhibitor(DM, Dorsomorphin) to the otic vesicle can partically rescue the semicircular canals phenotype.In summary, Imo4a in vivo may regulate Bmps, promoting semicircular morphogenesis. This is the first study exploring a significant opportunity for the development of local delivery of the small molecule DM as a totally new therapeutic approach for inner ear malformation and deafness.更多还原