耳蜗突触带的发育观察和长期中等强度噪声暴露耳蜗损伤的机制研究

发布时间：2018-03-24 08:40

  本文选题：突触带　切入点：发育　出处：《南方医科大学》2017年硕士论文

【摘要】：目的了解耳蜗突触带发育的时间特点和空间分布,明确C57BL/6j小鼠突触带稳定的时间,探讨长期中等强度噪声暴露耳蜗损伤的病理机制。方法(1)全耳蜗基底膜铺片、免疫荧光染色后,通过共聚焦显微镜观察、记录不同发育阶段耳蜗毛细胞突触前膜上突触带的发育与分布;(2)应用免疫荧光、HE染色、耳蜗电镜扫描评估耳蜗除带状突触之外的结构成分的变化;(3)中等强度白噪声,70dBSPL,8h/天,持续暴露3月,建造模拟环境噪声慢性暴露的动物模型;(4)采用ABR检测评估听觉阈值和诱发电位Ⅰ波幅度;(5)应用小鼠耳蜗音频定位图,建立全耳蜗突触带与听觉频谱之间的对应关系。结果(1)C57小鼠出生后Od,在内外毛细胞的胞质即可观察到CtBP2的信号表达;出生后第6天,CtBP2的信号数目在内外毛细胞的胞质和基底侧膜上的表达均达到高峰,突触带信号的增长在外毛细胞上则表现得尤为明显;出生后第12-60d,内外毛细胞上的CtBP2信号数目急剧减少并逐渐趋于稳定,且在毛细胞上的分布逐渐向基底侧膜聚集。功能学上,ABR可检测听力出现于出生后13.2天;(2)内毛细胞上突触带富集于耳蜗中间区域(距顶转50%-70%),可达18.6个/细胞,明显高于突触带在耳蜗顶转10%-20%(7.4个/细胞)和底转90%-100%(9.6个/细胞)区域的分布。外毛细胞沿耳蜗基底膜螺旋分布未见明显差异,1-3个/细胞;(3)中等强度噪声暴露3个月,在听觉频谱范围内,各频段均有不同程度的听力减退,且以8-16kHz频段阈值及ABRⅠ波幅度下降明显;(4)借助耳蜗音频定位,突触带计数显示,沿耳蜗螺旋突触带数目广泛减少,且在距耳蜗顶转50-70%区域突触带减少明显高于其他区域。(5)与对照组相比,毛细胞计数、静纤毛排列、螺旋神经元计数未见明显差异。结论(1)幼年C57小鼠,毛细胞与蜗神经纤维的突触联系出生前即已存在。出生后12d前,突触带经历数目和空间分布上的剧烈变化,12d后逐渐趋于完善和功能上的成熟;出生13.2d后,小鼠开始出现听力。因此,突触带成熟的时间和小鼠听觉发生时间的吻合,提示功能成熟的突触带是听觉发生的前提之一;(2)60日龄的C57小鼠,突触带沿耳蜗的分布趋于稳定;(3)长期中等强度噪声暴露可致在多个频率上不同程度的听阈提高,且暴露对听觉敏感区(8-16kHz)影响最为显著,其潜在的病理机制可能是原发性的内毛细胞与听觉传入纤维连接的大量减少所致。
[Abstract]:Objective to understand the distribution characteristics of time and space with the development of cochlear synapses, clear synaptic C57BL/6j mice with stable time, to investigate the pathologic mechanism of long-term moderate intensity noise exposure in cochlear injury. Methods (1) the basilar membrane preparations, immunofluorescence staining, confocal microscopy through recording, development and distribution of different synaptic ribbon developmental stages of cochlear hair cells in the presynaptic membrane; (2) by immunofluorescence, HE staining, scanning electron microscopy evaluation of cochlear structure changes of cochlear ingredients except outside the ribbon synapse; (3) moderate intensity white noise, 70dBSPL, 8h/ days, continuous exposure in March, the animal model of chronic exposure to environmental noise building simulation; (4) using ABR detection and evaluation of auditory threshold and evoked potential amplitude of 1; (5) using the mouse cochlear audio location map, establish the whole cochlear and auditory synapse with spectral correspondence between results. (1) C57 mice Od , to CtBP2 signal was observed on the inner and outer hair cells in the cytoplasm can be expressed; sixth days after birth, the number of signals in the CtBP2 expression of outer hair cell cytoplasm and the basolateral membrane of synaptic growth reached the peak, band signals in outer hair cells is particularly evident; 12-60d after birth. The number of CtBP2 signal on the inner and outer hair cells decreased sharply and gradually stabilized, and the distribution in the hair cells gradually to the basolateral membrane aggregation. Functionally, ABR can detect the hearing in 13.2 days after birth; (2) the inner hair cells on the synaptic ribbon enriched in the region in the cochlea (between from the top to 50%-70%), up to 18.6 per cell, was significantly higher than that of 10%-20% in the apical synaptic ribbon (7.4 / cell) and bottom 90%-100% (9.6 / cell) region. The distribution of the outer hair cells along the basilar membrane of the cochlea spiral distribution showed no significant difference, 1-3 / cells; (3) the medium intensity noise exposure 3 April, in the auditory frequency range, the band had different degrees of hearing loss, and to the 8-16kHz band and ABR 1 threshold amplitude decreased significantly; (4) with the aid of cochlear audio localization, synaptic counting display, along the spiral belt and reduce the number of synapses widely, with reduced significantly higher than other regions from the apical turn 50-70% synapses. (5) compared with the control group, hair cell counting, stereocilia arranged, spiral neurons count showed no significant difference. Conclusion (1) in young C57 mice, synaptic connections between hair cells and cochlear nerve fibers before birth has saved in. After the birth of 12D, with changing the number and synaptic space the distribution of 12D tends to improve gradually and functionally mature; born after 13.2d, the mice started listening. Therefore, take time to mature mice and synaptic auditory time with synaptic functional maturation with auditory occurrence One of the premises; (2) the 60 day old C57 mice, with synaptic distribution along the cochlea tends to be stable; (3) long-term medium intensity noise exposure can cause at multiple frequencies with different degrees of exposure on the auditory threshold was increased, and the sensitive area (8-16kHz) has the most significant impact, the underlying pathogenesis may be primary inner hair cells and auditory afferent fibers connecting the large decrease.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R764.43
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本文编号：1657541