噪声引起的耳蜗金属蛋白酶变化及其对听功能的影响

发布时间：2018-04-30 15:40

本文选题：噪声性耳聋 + 活性氮　；参考：《中国人民解放军军医进修学院》2013年硕士论文

【摘要】：噪声造成听觉系统永久性损害，是最常见的职业性伤害和听力致残因素，给社会及个人带来沉重的经济和精神负担。因此,如何预防噪声性耳聋(NIHL)的发生和降低听觉损害的程度是耳科学者的重要课题。已有研究表明噪声引起耳蜗损伤是多方面的,包括机械损伤、耳蜗微循环的改变导致的血流减少、代谢紊乱导致毛细胞的死亡以及耳蜗外侧壁血迷路屏障通透性改变等。噪声暴露后引起耳蜗内代谢改变及其引起的一系列变化被认为是造成不可逆的感音神经性耳聋的关键因素，但其具体机制尚不十分明确。故在本课题中,我们研究噪声引起的耳蜗活性氮自由基（RNS）及金属基质蛋白酶(MMPs)的变化及其对听功能的影响。结果如下：一、噪声引起的耳蜗氮自由基变化及其对听功能的影响 NO介导的细胞病理性反应主要是通过迅速氧化为过氧亚硝基阴离子（Peroxynitrite ONOO-）来实现，ONOO-是最具活性及损害力的一种活性氮自由基，可以造成细胞蛋白质、核酸及脂质膜的损伤，最终触发细胞凋亡并且能够直接或间接的破坏机体屏障系统。ONOO-通过硝基化酪氨酸及其残体而发挥作用，，在这一过程中会产生3-硝基酪氨酸（3-nitrotyrosine3-NT）。本课题通过3-NT作为检测活性氮自由基的标志物。在成功建立120dBSPL白噪声致聋模型的基础上，通过听性脑干反应、畸变耳声发射、电诱发听性脑干反应等听觉生理指标评估该模型的听力损失程度，发现噪声暴露后ABR阈值、eABR刺激幅值均有显著的增加，DPOAE波幅显著下降，说明120dBSPL白噪声建立的动物模型具有稳定的听力损失，且在外毛细胞及听神经均有损伤。利用耳蜗铺片及颞骨冰冻切片、免疫组织化学、激光共聚焦显微镜扫描发现：正常情况下，耳蜗内也有RNS的存在，在耳蜗毛细胞主要分布于胞质内及表皮板，而细胞核及其周围很少分布,在耳蜗外侧壁的血管纹上及螺旋神经节也有RNS分布；噪声暴露后耳蜗毛细胞、血管纹及螺旋神经节上标记ONOO-的荧光明显增强，说明噪声刺激下耳蜗内RNS在毛细胞、血管纹及螺旋神经节含量增加，为了进一步定量证实上述结果，通过Western blot检测技术结果显示噪声暴露后耳蜗内RNS含量明显升高。二、噪声引起的耳蜗金属蛋白酶改变及其对听功能的影响有研究表明，ONOO－在脑缺血损伤中能够激活基质金属蛋白酶(MMPs)，进一步降解脑血管基膜上的紧密连接蛋白，从而破坏血脑屏障(BBB)。血迷路屏障亦是如血脑屏障一样的人体屏障系统，噪声暴露能否引起MMPs的增加从而破坏血迷路屏障正是本部分实验所探讨的问题。MMP-9和MMP-2是耳蜗内分布的主要的MMPs家族成员，是唯一能够降解耳蜗中细胞外基质的蛋白水解酶。正常含量的MMPs是有益的，但是在损伤致明显上调后是有害的。从理论上讲，MMPs在耳蜗受到损伤后可能被激活从而使血迷路屏障通透性增加，K+浓度改变，扰乱了内淋巴的稳态，导致感音神经性聋。本部分实验在第一部分成功建立噪声性耳聋动物模型的基础上，利用耳蜗铺片及颞骨冰冻切片、免疫组织化学、激光共聚焦显微镜扫描等技术，研究发现：正常情况下，耳蜗内也有MMPs的存在，MMP-9主要分布在耳蜗外侧壁血管纹上，MMP-2主要在耳蜗基底膜分布；噪声暴露后耳蜗内标记MMP-9，MMP-2的荧光明显增强，并通过Western blot检测技术从分子生物学角度进一步定量验证了噪声损伤能诱使MMP-9，MMP-2含量增加，推测噪声造成的听力损失与MMPs增多降解基底膜和耳蜗外侧壁血管纹的细胞外基质从而造成了内环境紊乱有关。本实验初步在细胞及亚细胞水平探寻噪声性耳聋的分子生物学机制，研究结果显示噪声暴露能够导致耳蜗内活性氮自由基及MMPs升高，且对听功能产生显著影响。由此阐述此代谢变化造成的耳蜗病理损伤的机制。从噪声性耳聋发病的细胞及分子学机制角度，为寻找预防和治疗噪声性耳聋这一难题开辟了新的途径。
[Abstract]:Noise causes permanent damage to the auditory system. It is the most common occupational injury and hearing loss factor, which brings a heavy economic and spiritual burden to the society and the individual. Therefore, how to prevent the occurrence of noise induced deafness (NIHL) and reduce the degree of hearing impairment is an important topic for the ear scholars. There are many aspects, including mechanical damage, the decrease of blood flow caused by changes in the cochlear microcirculation, the death of the hair cell and the change of the permeability of the labyrinth barrier in the outer wall of the cochlea. The changes in the internal metabolism of the cochlea and a series of changes in the cochlea caused by noise exposure are considered to cause irreversible sensorineural deafness. Key factors, but their specific mechanisms are not yet very clear. So in this subject, we study the changes in nitrogen free radicals (RNS) and metal matrix protease (MMPs) induced by noise in the cochlea and their effects on the auditory function.
1. Noise induced changes of nitrogen free radicals in cochlea and their effects on hearing function.
The cytopathic reaction mediated by NO is mainly achieved by rapid oxidation to the peroxy nitroso anion (Peroxynitrite ONOO-). ONOO- is the most active and damaging active nitrogen radical, which can cause damage to the cell protein, nucleic acid and lipid membrane, eventually triggers cell apoptosis and can destroy the body directly or indirectly. The barrier system.ONOO- plays a role in the nitration of tyrosine and its residues and produces 3- nitro tyrosine (3-nitrotyrosine3-NT) in this process. This subject uses 3-NT as a marker for detecting the free radicals of active nitrogen. On the basis of the successful establishment of the 120dBSPL white noise induced deafness model, the auditory brainstem response is used to distort the otoacoustic emission, The hearing loss of the model was evaluated by auditory physiological indexes such as auditory brainstem response. It was found that the threshold of ABR after noise exposure, the amplitude of eABR stimulation had a significant increase, and the amplitude of DPOAE decreased significantly. It indicated that the animal model established by the white noise of 120dBSPL had stable hearing loss and had damage in both the outer hair cell and the auditory nerve. The cochlear slice and the frozen section of the temporal bone, immunohistochemistry and laser confocal microscope scan found that under normal conditions, there were also RNS in the cochlea, and the cochlear hair cells were mainly distributed in the cytoplasm and the epidermis, but the nucleus and its surrounding were rarely distributed, and there were RNS distribution on the veins of the lateral wall of the cochlea and the spiral ganglion. The fluorescence of the cochlear hair cells, vascular lines and spiral ganglion labeled ONOO- increased significantly after noise exposure, indicating that the content of RNS in the hair cells, vascular lines and spiral ganglia in the cochlea increased under noise stimulation. In order to further confirm the above results, the RNS content in the cochlea of the cochlea after noise exposure was revealed by the Western blot detection technique. Increase significantly.
Two, noise induced changes in cochlear metalloproteinases and their effects on hearing function.
Some studies have shown that oto can activate matrix metalloproteinases (MMPs) in cerebral ischemia injury, further degrade the close connexin on the cerebral vascular basement membrane, and destroy the blood brain barrier (BBB). The blood labyrinth barrier is also the human barrier system like the blood brain barrier, and whether the noise exposure can cause the increase of MMPs and destroy the blood labyrinth barrier .MMP-9 and MMP-2 are the main MMPs family members of the inner cochlea, the only protein hydrolase that can degrade the extracellular matrix in the cochlea. The MMPs of the normal content is beneficial, but it is harmful after the damage is obviously up-regulated. In theory, MMPs may be stimulated after the cochlea is damaged. On the basis of the successful establishment of a noise induced deafness animal model, this part of the experiment was conducted by using the cochlear sheet and the frozen section of the temporal bone, immunohistochemistry, laser scanning confocal microscopy and so on. It was found that, under normal conditions, there was MMPs in the cochlea, and MMP-9 was mainly distributed in the veins of the lateral wall of the cochlea. MMP-2 was mainly distributed in the cochlear basement membrane. After the noise exposure, the cochlea was marked with MMP-9, and the fluorescence of MMP-2 was obviously enhanced. The noise damage energy was further verified by the Western blot detection technique from the molecular biological angle. The increase in the content of MMP-9, MMP-2, and the inference of the hearing loss caused by noise are related to the increasing degradation of the extracellular matrix of the basal membrane and the lateral wall of the cochlea by increasing MMPs.
In this experiment, the molecular biological mechanism of noise induced deafness was explored at the cellular and subcellular level. The results showed that noise exposure could lead to the increase of active nitrogen free radicals and MMPs in the cochlea, and had a significant effect on the auditory function. From the point of view of cell and molecular mechanism, it opens up a new way to find out the problem of prevention and treatment of noise induced hearing loss.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R764.43

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