發(fā)布時間：2018-04-30 15:40

  本文選題：噪聲性耳聾 + 活性氮　； 參考：《中國人民解放軍軍醫(yī)進修學院》2013年碩士論文

【摘要】：噪聲造成聽覺系統(tǒng)永久性損害，是最常見的職業(yè)性傷害和聽力致殘因素，給社會及個人帶來沉重的經(jīng)濟和精神負擔。因此,如何預防噪聲性耳聾(NIHL)的發(fā)生和降低聽覺損害的程度是耳科學者的重要課題。已有研究表明噪聲引起耳蝸損傷是多方面的,包括機械損傷、耳蝸微循環(huán)的改變導致的血流減少、代謝紊亂導致毛細胞的死亡以及耳蝸外側壁血迷路屏障通透性改變等。噪聲暴露后引起耳蝸內代謝改變及其引起的一系列變化被認為是造成不可逆的感音神經(jīng)性耳聾的關鍵因素，但其具體機制尚不十分明確。故在本課題中,我們研究噪聲引起的耳蝸活性氮自由基（RNS）及金屬基質蛋白酶(MMPs)的變化及其對聽功能的影響。結果如下： 一、噪聲引起的耳蝸氮自由基變化及其對聽功能的影響 NO介導的細胞病理性反應主要是通過迅速氧化為過氧亞硝基陰離子（Peroxynitrite ONOO-）來實現(xiàn)，ONOO-是最具活性及損害力的一種活性氮自由基，可以造成細胞蛋白質、核酸及脂質膜的損傷，最終觸發(fā)細胞凋亡并且能夠直接或間接的破壞機體屏障系統(tǒng)。ONOO-通過硝基化酪氨酸及其殘體而發(fā)揮作用，，在這一過程中會產(chǎn)生3-硝基酪氨酸（3-nitrotyrosine3-NT）。本課題通過3-NT作為檢測活性氮自由基的標志物。在成功建立120dBSPL白噪聲致聾模型的基礎上，通過聽性腦干反應、畸變耳聲發(fā)射、電誘發(fā)聽性腦干反應等聽覺生理指標評估該模型的聽力損失程度，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，說明120dBSPL白噪聲建立的動物模型具有穩(wěn)定的聽力損失，且在外毛細胞及聽神經(jīng)均有損傷。利用耳蝸鋪片及顳骨冰凍切片、免疫組織化學、激光共聚焦顯微鏡掃描發(fā)現(xiàn)：正常情況下，耳蝸內也有RNS的存在，在耳蝸毛細胞主要分布于胞質內及表皮板，而細胞核及其周圍很少分布,在耳蝸外側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胞、血管紋及螺旋神經(jīng)節(jié)上標記ONOO-的熒光明顯增強，說明噪聲刺激下耳蝸內RNS在毛細胞、血管紋及螺旋神經(jīng)節(jié)含量增加，為了進一步定量證實上述結果，通過Western blot檢測技術結果顯示噪聲暴露后耳蝸內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聽功能的影響 有研究表明，ONOO－在腦缺血損傷中能夠激活基質金屬蛋白酶(MMPs)，進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驗所探討的問題。MMP-9和MMP-2是耳蝸內分布的主要的MMPs家族成員，是唯一能夠降解耳蝸中細胞外基質的蛋白水解酶。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，擾亂了內淋巴的穩(wěn)態(tài)，導致感音神經(jīng)性聾。本部分實驗在第一部分成功建立噪聲性耳聾動物模型的基礎上，利用耳蝸鋪片及顳骨冰凍切片、免疫組織化學、激光共聚焦顯微鏡掃描等技術，研究發(fā)現(xiàn)：正常情況下，耳蝸內也有MMPs的存在，MMP-9主要分布在耳蝸外側壁血管紋上，MMP-2主要在耳蝸基底膜分布；噪聲暴露后耳蝸內標記MMP-9，MMP-2的熒光明顯增強，并通過Western blot檢測技術從分子生物學角度進一步定量驗證了噪聲損傷能誘使MMP-9，MMP-2含量增加，推測噪聲造成的聽力損失與MMPs增多降解基底膜和耳蝸外側壁血管紋的細胞外基質從而造成了內環(huán)境紊亂有關。 本實驗初步在細胞及亞細胞水平探尋噪聲性耳聾的分子生物學機制，研究結果顯示噪聲暴露能夠導致耳蝸內活性氮自由基及MMPs升高，且對聽功能產(chǎn)生顯著影響。由此闡述此代謝變化造成的耳蝸病理損傷的機制。從噪聲性耳聾發(fā)病的細胞及分子學機制角度，為尋找預防和治療噪聲性耳聾這一難題開辟了新的途徑。
[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.

【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R764.43

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