白化荣昌猪耳聋的分子病理机制研究

发布时间：2018-03-12 21:48

本文选题：Mitf-M基因　切入点：基因突变　出处：《中国人民解放军医学院》2013年博士论文　论文类型：学位论文

【摘要】：小眼畸形相关转录因子（Mitf）是由Mitf基因编码的一个拥有碱性螺旋-环-螺旋拉链（basic helix-loop-helix zipper，bHLH-Zip)结构的转录因子[1]。Mitf调控着许多细胞的分化，如黑色素细胞，视杯来源的视网膜色素上皮细胞（RPE）以及许多类型的骨髓来源的细胞[2]。根据氨基末端的不同，Mitf包含至少5种基因亚型，每种亚型的启动子以及起始外显子都不同，，并且表达在不同的组织中，而Mitf-M特异性表达在黑色素细胞和黑色素瘤细胞中[3-7]。人类的Waardenburg综合征是一种以听觉-色素异常为特点的常染色体显性遗传疾病，占先天性耳聋的2%[8]。按临床特征和遗传标准共分为四种亚型。其中Waardenburg综合征2A型及其严重类型Tietz综合征是由MITF-M基因突变引起的，表现为感音神经性耳聋、虹膜异色和白色额发[9]。尽管大量研究已经证明Mitf-M基因突变将导致神经嵴来源的黑色素细胞缺失，最终产生耳聋等症状，但是Mitf基因对听觉系统的作用及其分子病理机制至今未明[10]。因此，仔细研究此亚型Mitf基因的结构和功能对于耳聋的基因诊断和分子病理机制研究是很重要的。本研究通过建立正常荣昌猪内耳解剖、手术方式以及形态、听功能数据库，发现猪的内耳和人类及其他动物相比在功能和形态上既具有相似性又存在差异，为下一步研究白化耳聋荣昌猪奠定了基础；通过对白化耳聋荣昌猪进行家系构建和基因分析，确定精确的致病基因为Mitf-M基因，使得此耳聋动物家系遗传背景清楚，可以作为耳聋疾病的大型哺乳动物模型应用于耳科领域的研究；利用从胚胎到出生后不同发育阶段的自发突变型白化荣昌猪（Mitf-/-）和正常野生型荣昌猪（Mitf+/+）内耳形态学和听功能的比较分析，发现白化荣昌猪的耳聋是由Mitf-M基因突变导致内耳血管纹病理变化引起的，最终引发血管纹中间细胞消失，内耳钾离子浓度和耳蜗内电位的变化，导致ABR异常，听功能丧失，阐述了Mitf-M基因突变引发耳聋的病理机制。使我们进一步明确了胚胎发育过程中Mitf-M对血管纹和黑色素细胞发生作用的关键时间点和变化规律。这可能也是人类Waardenburg综合征2A型及Tietz综合征所致耳聋症状的原因。为研究人类Waardenburg综合征2A型感音神经性耳聋的早期基因干预治疗、人工听力重建、毛细胞再生等提供了重要的基础数据，为人类Waardenburg综合征2A型耳聋的研究提供了理想的大型哺乳动物模型。本研究的重要发现及其意义： 1.首次报道了正常荣昌猪内耳超微结构； 2.首次发现了白化荣昌猪的耳聋表型与人类Waardenburg综合征2A型Mitf-M基因突变导致的耳聋表型一致； 3.首次报道了Mitf-M基因突变与白化荣昌猪内耳血管纹病理变化的关系； 4.揭示了Waardenburg综合征2A型发病的分子病理机制。
[Abstract]:Microocular deformation-associated transcription factor (Mitf) is a transcription factor with basic helix-loop-helix zipperbHLH-Zipp structure encoded by the Mitf gene. [1] .Mitf regulates the differentiation of many cells, such as melanocytes. Retinal pigment epithelial cells (RPEs) and many types of bone marrow-derived cells [2]. Different amino terminal mitf contains at least five gene subtypes, each of which has different promoters and initial exons. And expressed in different tissues, while Mitf-M was specifically expressed in melanocytes and melanoma cells [3-7]. Human Waardenburg syndrome is an autosomal dominant genetic disease characterized by acoustic-pigment abnormalities. 2% patients with congenital deafness were divided into four subtypes according to clinical characteristics and genetic criteria. Type 2A of Waardenburg syndrome and its severe type Tietz syndrome were caused by mutation of MITF-M gene and presented as sensorineural deafness. Iris heterochromatic and white frontalis [9] .Although a large number of studies have shown that mutations in the Mitf-M gene can lead to the deletion of melanocytes from neural crest, resulting in deafness and other symptoms, However, the role of Mitf gene in the auditory system and its molecular pathological mechanism have not been clarified. Therefore, it is important to study the structure and function of this subtype of Mitf gene for gene diagnosis and molecular pathological mechanism of deafness. In this study, we established the normal Rong Chang pig inner ear anatomy, operation method and shape, auditory function database, found that pig inner ear compared with human and other animals in the function and morphology of both similarities and differences. It lays a foundation for the further study of Rongchang pig with albino deafness, and through the family construction and gene analysis of Rongchang pig with albino deafness, it is determined that the exact pathogenic gene is Mitf-M gene, which makes the genetic background of this deafness animal family clear. It can be used as a large mammal model of deafness disease in the field of otology. The inner ear morphology and auditory function of Mitf-r / -) and Mitf /) of spontaneous mutant Rongchang albino from embryo to postnatal development stage were compared and analyzed. It was found that the deafness of Rongchang pig was caused by the pathological changes of the vascular stria of the inner ear caused by the mutation of Mitf-M gene, and the disappearance of the middle cell of the stria vascularis, the change of the concentration of potassium ion in the inner ear and the change of the potential in the cochlea, which resulted in the abnormality of ABR and the loss of auditory function. The pathological mechanism of deafness caused by mutation of Mitf-M gene was expounded. The key time point and change rule of Mitf-M on stria vascularis and melanocytes during embryonic development were further clarified. This may also be the ensemble of human Waardenburg. In order to study the early gene intervention therapy of human Waardenburg syndrome type 2A sensorineural deafness, the causes of deafness caused by type 2A syndromes and Tietz syndrome were studied. Artificial hearing reconstruction and hair cell regeneration provide important data for the study of human Waardenburg syndrome type 2A deafness and provide an ideal large mammal model. The important findings of this study and its significance:. 1. The ultrastructure of the inner ear of the normal Rongchang pig was reported for the first time. 2. The deafness phenotype of Rongchang albino pig was found to be consistent with the deafness phenotype caused by the mutation of Mitf-M gene of human Waardenburg syndrome type 2A for the first time. 3. The relationship between the mutation of Mitf-M gene and the pathological changes of vascular stria in the inner ear of Rongchang pig was reported for the first time. 4. The molecular pathological mechanism of type 2A of Waardenburg syndrome was revealed.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R764.43

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