本文選題：噪聲性耳聾 + 活性氮��； 參考：《中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院》2013年碩士論文

【摘要】：噪聲造成聽(tīng)覺(jué)系統(tǒng)永久性損害，是最常見(jiàn)的職業(yè)性傷害和聽(tīng)力致殘因素，給社會(huì)及個(gè)人帶來(lái)沉重的經(jīng)濟(jì)和精神負(fù)擔(dān)。因此,如何預(yù)防噪聲性耳聾(NIHL)的發(fā)生和降低聽(tīng)覺(jué)損害的程度是耳科學(xué)者的重要課題。已有研究表明噪聲引起耳蝸損傷是多方面的,包括機(jī)械損傷、耳蝸微循環(huán)的改變導(dǎo)致的血流減少、代謝紊亂導(dǎo)致毛細(xì)胞的死亡以及耳蝸外側(cè)壁血迷路屏障通透性改變等。噪聲暴露后引起耳蝸內(nèi)代謝改變及其引起的一系列變化被認(rèn)為是造成不可逆的感音神經(jīng)性耳聾的關(guān)鍵因素，但其具體機(jī)制尚不十分明確。故在本課題中,我們研究噪聲引起的耳蝸活性氮自由基（RNS）及金屬基質(zhì)蛋白酶(MMPs)的變化及其對(duì)聽(tīng)功能的影響。結(jié)果如下： 一、噪聲引起的耳蝸氮自由基變化及其對(duì)聽(tīng)功能的影響 NO介導(dǎo)的細(xì)胞病理性反應(yīng)主要是通過(guò)迅速氧化為過(guò)氧亞硝基陰離子（Peroxynitrite ONOO-）來(lái)實(shí)現(xiàn)，ONOO-是最具活性及損害力的一種活性氮自由基，可以造成細(xì)胞蛋白質(zhì)、核酸及脂質(zhì)膜的損傷，最終觸發(fā)細(xì)胞凋亡并且能夠直接或間接的破壞機(jī)體屏障系統(tǒng)。ONOO-通過(guò)硝基化酪氨酸及其殘?bào)w而發(fā)揮作用，，在這一過(guò)程中會(huì)產(chǎn)生3-硝基酪氨酸（3-nitrotyrosine3-NT）。本課題通過(guò)3-NT作為檢測(cè)活性氮自由基的標(biāo)志物。在成功建立120dBSPL白噪聲致聾模型的基礎(chǔ)上，通過(guò)聽(tīng)性腦干反應(yīng)、畸變耳聲發(fā)射、電誘發(fā)聽(tīng)性腦干反應(yīng)等聽(tīng)覺(jué)生理指標(biāo)評(píng)估該模型的聽(tīng)力損失程度，發(fā)現(xiàn)噪聲暴露后ABR閾值、eABR刺激幅值均有顯著的增加，DPOAE波幅顯著下降，說(shuō)明120dBSPL白噪聲建立的動(dòng)物模型具有穩(wěn)定的聽(tīng)力損失，且在外毛細(xì)胞及聽(tīng)神經(jīng)均有損傷。利用耳蝸鋪片及顳骨冰凍切片、免疫組織化學(xué)、激光共聚焦顯微鏡掃描發(fā)現(xiàn)：正常情況下，耳蝸內(nèi)也有RNS的存在，在耳蝸毛細(xì)胞主要分布于胞質(zhì)內(nèi)及表皮板，而細(xì)胞核及其周?chē)苌俜植?在耳蝸外側(cè)壁的血管紋上及螺旋神經(jīng)節(jié)也有RNS分布；噪聲暴露后耳蝸毛細(xì)胞、血管紋及螺旋神經(jīng)節(jié)上標(biāo)記ONOO-的熒光明顯增強(qiáng)，說(shuō)明噪聲刺激下耳蝸內(nèi)RNS在毛細(xì)胞、血管紋及螺旋神經(jīng)節(jié)含量增加，為了進(jìn)一步定量證實(shí)上述結(jié)果，通過(guò)Western blot檢測(cè)技術(shù)結(jié)果顯示噪聲暴露后耳蝸內(nèi)RNS含量明顯升高。 二、噪聲引起的耳蝸金屬蛋白酶改變及其對(duì)聽(tīng)功能的影響 有研究表明，ONOO－在腦缺血損傷中能夠激活基質(zhì)金屬蛋白酶(MMPs)，進(jìn)一步降解腦血管基膜上的緊密連接蛋白，從而破壞血腦屏障(BBB)。血迷路屏障亦是如血腦屏障一樣的人體屏障系統(tǒng)，噪聲暴露能否引起MMPs的增加從而破壞血迷路屏障正是本部分實(shí)驗(yàn)所探討的問(wèn)題。MMP-9和MMP-2是耳蝸內(nèi)分布的主要的MMPs家族成員，是唯一能夠降解耳蝸中細(xì)胞外基質(zhì)的蛋白水解酶。正常含量的MMPs是有益的，但是在損傷致明顯上調(diào)后是有害的。從理論上講，MMPs在耳蝸受到損傷后可能被激活從而使血迷路屏障通透性增加，K+濃度改變，擾亂了內(nèi)淋巴的穩(wěn)態(tài)，導(dǎo)致感音神經(jīng)性聾。本部分實(shí)驗(yàn)在第一部分成功建立噪聲性耳聾動(dòng)物模型的基礎(chǔ)上，利用耳蝸鋪片及顳骨冰凍切片、免疫組織化學(xué)、激光共聚焦顯微鏡掃描等技術(shù)，研究發(fā)現(xiàn)：正常情況下，耳蝸內(nèi)也有MMPs的存在，MMP-9主要分布在耳蝸外側(cè)壁血管紋上，MMP-2主要在耳蝸基底膜分布；噪聲暴露后耳蝸內(nèi)標(biāo)記MMP-9，MMP-2的熒光明顯增強(qiáng)，并通過(guò)Western blot檢測(cè)技術(shù)從分子生物學(xué)角度進(jìn)一步定量驗(yàn)證了噪聲損傷能誘使MMP-9，MMP-2含量增加，推測(cè)噪聲造成的聽(tīng)力損失與MMPs增多降解基底膜和耳蝸外側(cè)壁血管紋的細(xì)胞外基質(zhì)從而造成了內(nèi)環(huán)境紊亂有關(guān)。 本實(shí)驗(yàn)初步在細(xì)胞及亞細(xì)胞水平探尋噪聲性耳聾的分子生物學(xué)機(jī)制，研究結(jié)果顯示噪聲暴露能夠?qū)е露亙?nèi)活性氮自由基及MMPs升高，且對(duì)聽(tīng)功能產(chǎn)生顯著影響。由此闡述此代謝變化造成的耳蝸病理?yè)p傷的機(jī)制。從噪聲性耳聾發(fā)病的細(xì)胞及分子學(xué)機(jī)制角度，為尋找預(yù)防和治療噪聲性耳聾這一難題開(kāi)辟了新的途徑。
[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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R764.43

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