lmo4a基因在斑马鱼内耳发育中的功能研究

发布时间：2018-09-10 16:04

【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。
[Abstract]:Objective: there are about 278 million people with moderate or more hearing loss worldwide and 27.8 million people with hearing and speech disability in China. The prevention and treatment of deafness mainly include conductive deafness and sensorineural deafness. The prevention and treatment of sensorineural deafness has been a hot and difficult point in the basic research of deafness since half a century, and the prevention and treatment of sensorineural deafness have gained mature experience. Clinically, many sensorineural deafness is caused by abnormal development of the inner ear. The course of inner ear deformity and deafness is complicated, the function is irreversible, and the treatment method is relatively limited. Searching for more genes causing deafness and deafness, exploring the mechanism of deafness, and screening specific therapeutic agents for the pathogenesis of deafness have become the common responsibility of the basis of otorology and clinical researchers to encode a transcriptional regulatory factor of .LM04 gene. The abnormal development of semicircular canal and dysplasia of semicircular canal cristae were observed in LM04, with tissue specific knockout in mouse ear vesicles. So LM04 gets more scholars' attention. Zebrafish internal ear formation mechanism, vestibular morphology, conserved function, transparent embryo, easy to operate, is the dominant model for the study of deformity and deafness of the inner ear. In this study, the effect of lmo4 gene on the development of zebrafish inner ear was studied, and the mechanism was explored to lay a theoretical foundation for the prevention and treatment of deformity of inner ear and sensorineural deafness. Methods: a low expression and overexpression model of lmo4 gene was constructed by injecting oligonucleotide Morpholino (MO) and mRNA into zebrafish embryos. The important role of lmo4 gene in the development of inner ear of zebrafish was studied by immunohistochemistry, and the mechanism of lmo4 gene affecting the development of inner ear was studied by screening the gene expression changes closely related to the structure of inner ear. And to screen the mechanism of the treatment of phenotypic agents. Results: the two homologous genes of lmo4 in zebrafish, lmo4a and lmo4b, were both maternal genes. 10hpflmo4a was mainly expressed in mesoderm and nerve plate. With the development of auricle, the expression range of lmo4a overlapped with that of hair cells, and the expression of three semicircular canal crest and semicircular canal protrusions, which were closely related to semicircular canal development, was continuously expressed in auricle, peripheral matrix and lateral line. No phenotype of inner ear was observed after low expression of lmo4b in Morpholino. Our later studies focused on the function of lmo4a in inner ear. After the low expression of lmo4a in Morpholino, it was found that the development of anterior basal plate was blocked, the size of ear follicles, the number of hair cells and the number of cells in auditory balanced ganglion decreased, and the abnormal development of semicircular canal (only horizontal semicircular canal processes) was observed. Bmp4,bmp2b closely related to semicircular canal development was upregulated, and down-regulation of BMP signal could save the dysplastic phenotype of semicircular canal induced by low expression of lmo4a to a great extent. Conclusion: the decrease in the number of hair cells and ganglion of inner ear induced by low expression of lmo4a can be attributed to the effect of lmo4a on the early development of anterior basal plate, while the semicircular canal malformation is caused by upregulation of BMP signal. Intravesicular injection of DM (AMP-activated protein kinase inhibitor, Dorsomorphin) small molecule) can down-regulate BMP signal, promote semicircular canal development and save malformation. In this study, we investigated the important role and mechanism of lmo4a gene in the development of zebrafish inner ear. Injection of small molecule DM into ear vesicle could promote semicircular canal development and save semicircular canal malformation.
【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】：2012
【分类号】：R764

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