【摘要】：目的:研究蛋氨酸對噪聲性耳聾小鼠聽力損失的預(yù)防與治療作用。通過動物模型來研究抗氧化劑蛋氨酸對實驗對象耳蝸組織在功能學(xué)、形態(tài)學(xué)、氧化應(yīng)激水平和凋亡水平以及連接蛋白26(Cx26)和連接蛋白30(Cx30)方面是否具有預(yù)防和治療效果,并為研究噪聲性耳聾的發(fā)病機(jī)制提供進(jìn)一步依據(jù)。方法:1建立噪聲性耳聾動物模型:選取健康40只雌性昆明小鼠,根據(jù)隨機(jī)分組原則分為(1)空白對照組(n=10)(2)噪聲組(n=10)(3)噪聲前給予蛋氨酸組(n=10)(4)噪聲后給予蛋氨酸組(n=10)。首先進(jìn)行昆明小鼠外耳、中耳及內(nèi)耳的檢查,以排除影響內(nèi)耳聽力功能的因素。(1)空白對照組:不給予噪聲處理,正常環(huán)境下生活,腹腔注射生理鹽水400mg/Kg,2次/天,連續(xù)3天;(2)噪聲組:噪聲前3天給予生理鹽水400mg/Kg腹腔注射,2次/天,連續(xù)3天,再給予100dB SPL白噪聲,連續(xù)3天,每天持續(xù)8小時;(3)噪聲前給予蛋氨酸組:噪聲前3天給予蛋氨酸溶液400mg/Kg腹腔注射,連續(xù)3天,2次/天,再給予100dB SPL白噪聲,連續(xù)3天,每天持續(xù)8小時;(4)噪聲后給予蛋氨酸組:給予100dB SPL白噪聲,連續(xù)3天,每天持續(xù)8小時,噪聲3天結(jié)束后給予蛋氨酸溶液400mg/Kg腹腔注射,連續(xù)3天,2次/天。2監(jiān)測噪聲暴露前后小鼠聽力功能變化:四組昆明小鼠在噪聲暴露前、噪聲暴露后即刻和噪聲暴露后第4天,分別都接受聽性腦干反應(yīng)(ABR)來評估聽力功能。3顯微水平下耳蝸結(jié)構(gòu)改變:通過耳蝸基底膜鋪片HE染色并在普通光學(xué)顯微鏡下觀察毛細(xì)胞損傷情況,再通過耳蝸基底膜鋪片Myosin-Ⅵ染色在激光共聚焦顯微鏡下觀察基底膜毛細(xì)胞損傷情況。4免疫組織化學(xué)方法:通過免疫組織化學(xué)方法,檢測氧化應(yīng)激產(chǎn)物4-羥基壬烯醛(4-HNE),以評價各組氧化應(yīng)激水平。5 TUNEL凋亡檢測:通過TUNEL凋亡方法,觀察細(xì)胞凋亡情況。6westernblot法:使用westernblot法檢測cx26和cx30兩種縫隙鏈接蛋白在各組中的表達(dá)情況。結(jié)果:1一般觀察結(jié)果:空白對照組小鼠正常生長,各項反應(yīng)均好,耳廓對聲音反應(yīng)正常;噪聲組小鼠不好動,耳廓對聲音反應(yīng)較遲鈍;噪聲前給予蛋氨酸組的小鼠正常生長,耳廓對聲音反應(yīng)較對照組差;噪聲后給予蛋氨酸組的小鼠正常生長,耳廓對聲音反應(yīng)較對照組差。2聽覺腦干反應(yīng)(abr)測試結(jié)果:實驗前各組動物abr閾值沒有統(tǒng)計學(xué)差異(p0.05),空白對照組小鼠abr閾值為(16.25±3.58)dbspl,噪聲組小鼠abr閾值為(16.00±3.37)dbspl,噪聲前給予蛋氨酸組的小鼠abr閾值為(16.00±2.69)dbspl,噪聲后給予蛋氨酸組的小鼠abr閾值為(15.75±2.90)dbspl;噪聲暴露后即刻:空白對照組小鼠abr閾值為(16.75±3.74)dbspl,噪聲組小鼠abr閾值為(58.75±6.80)dbspl,噪聲前給予蛋氨酸組的小鼠abr閾值為(52.75±9.01)dbspl,噪聲后給予蛋氨酸組的小鼠abr閾值為(60.00±5.14)dbspl,除空白對照組外,其余接受噪聲處理的三組小鼠噪聲暴露前后自身配對比較均有統(tǒng)計學(xué)差異(p0.05);噪聲暴露后第4天,空白對照組小鼠abr閾值為(16.50±3.16)dbspl,噪聲組小鼠abr閾值為(36.25±7.48)dbspl,噪聲前給予蛋氨酸組的小鼠abr閾值為(23.00±3.29)dbspl,噪聲后給予蛋氨酸組的小鼠abr閾值為(23.25±3.13)dbspl,除空白對照組外,其余三組小鼠兩兩比較,噪聲組與噪聲前、后給予蛋氨酸組間比較均有統(tǒng)計學(xué)差異(p0.05),而噪聲前給予蛋氨酸組與噪聲后給予蛋氨酸組之間比較無統(tǒng)計學(xué)差異(p0.05)。3基底膜鋪片he染色和myosin-Ⅵ染色結(jié)果:空白對照組耳蝸內(nèi)、外毛細(xì)胞分布整齊,細(xì)胞輪廓清晰,未發(fā)現(xiàn)變性等病理改變。噪聲組與空白對照組相比,耳蝸基底膜毛細(xì)胞出現(xiàn)較為明顯的缺失,底轉(zhuǎn)外毛細(xì)胞尤為明顯。噪聲前給予蛋氨酸組與空白對照組相比,耳蝸基底膜毛細(xì)胞出現(xiàn)散在的缺失,可見變性等病理改變。噪聲后給予蛋氨酸組與空白對照組相比,耳蝸基底膜毛細(xì)胞同樣出現(xiàn)了散在的缺失,主要出現(xiàn)在外毛細(xì)胞,內(nèi)毛細(xì)胞結(jié)構(gòu)較為完整。44-hne免疫組織化學(xué)染色結(jié)果:空白對照組小鼠耳蝸組織切片中,在基底膜、血管紋和螺旋側(cè)韌帶中沒有發(fā)現(xiàn)4-HNE的表達(dá);噪聲組耳蝸基底膜、血管紋和螺旋側(cè)韌帶中4-HNE表達(dá)較明顯;噪聲前給予蛋氨酸組和噪聲后給予蛋氨酸組中,發(fā)現(xiàn)基底膜、血管紋和螺旋側(cè)韌帶中有4-HNE的表達(dá),但均弱于噪聲組。5 TUNEL凋亡檢測結(jié)果:空白對照組實驗動物耳蝸組織切片TUNEL染色中耳蝸基底膜、血管紋和螺旋側(cè)韌帶中沒有發(fā)現(xiàn)凋亡細(xì)胞;而在噪聲組上述三個結(jié)構(gòu)中均發(fā)現(xiàn)凋亡細(xì)胞,并且表達(dá)明顯;噪聲前給予蛋氨酸組和噪聲后給予蛋氨酸組均出現(xiàn)了凋亡細(xì)胞,但凋亡細(xì)胞數(shù)量明顯少于噪聲組。6 Cx26和Cx30 Western blot檢測結(jié)果:噪聲組中Cx26和Cx30的表達(dá)明顯弱于空白對照組、噪聲前給予蛋氨酸組和噪聲后給予蛋氨酸組,而空白對照組中Cx26和Cx30的表達(dá)強(qiáng)于噪聲前給予蛋氨酸組和噪聲后給予蛋氨酸組,但噪聲前給予蛋氨酸組和噪聲后給予蛋氨酸組中Cx26和Cx30的表達(dá)沒有明顯差異。結(jié)論:1噪聲性耳聾主要損傷耳蝸Corti器的內(nèi)、外毛細(xì)胞,并且以底轉(zhuǎn)外毛細(xì)胞損傷為主,蛋氨酸對噪聲性耳聾造成的聽力下降和形態(tài)結(jié)構(gòu)破壞起到一定的保護(hù)作用。2噪聲通過活性氧和細(xì)胞凋亡導(dǎo)致噪聲性耳聾的出現(xiàn),而蛋氨酸在活性氧和細(xì)胞凋亡水平上有一定的保護(hù)作用。3縫隙連接蛋白Cx26和Cx30可能與噪聲性耳聾的致病機(jī)制相關(guān),并且蛋氨酸可保護(hù)Cx26和Cx30在噪聲下的表達(dá)。4噪聲前給予蛋氨酸組和噪聲后給予蛋氨酸組結(jié)果表明了蛋氨酸對噪聲性耳聾有預(yù)防和治療作用。
[Abstract]:Objective: To study the preventive and therapeutic effects of methionine on hearing loss in noise-induced deafness mice. The animal models were used to study whether methionine, an antioxidant, has preventive and therapeutic effects on the function, morphology, oxidative stress, apoptosis, connexin 26 (Cx26) and connexin 30 (Cx30) of the cochlear tissues of experimental subjects. Methods: 1. Establishing the animal model of noise-induced deafness: 40 healthy female Kunming mice were divided into (1) blank control group (n = 10) (2) noise group (n = 10) (3) methionine group (n = 10) (4) methionine group (n = 10) after noise. The outer ear, middle ear and inner ear of Kunming mice were examined to exclude the factors affecting the hearing function of the inner ear. (1) blank control group: no noise treatment, living in normal environment, intraperitoneal injection of normal saline 400 mg / kg, twice a day, for three consecutive days; (2) noise group: before noise, intraperitoneal injection of normal saline 400 mg / kg, twice a day, for three consecutive days, and then Give 100dB SPL white noise for 3 days, lasting 8 hours a day; (3) Before noise, give methionine group: before noise, give methionine solution 400mg/kg intraperitoneal injection for 3 days, twice a day, and then give 100dB SPL white noise for 3 days, lasting 8 hours a day; (4) after noise, give methionine group: give 100dB SPL white noise for 3 days, hold it daily for 3 days. After 3 days of noise exposure, the mice were injected with methionine solution 400mg/kg intraperitoneally for 8 hours. The changes of hearing function were monitored for 3 days, 2 times a day. Changes in the structure of the lower cochlea: The damage of hair cells was observed by HE staining on the basilar membrane of the cochlea and by Myosin-VI staining on the basilar membrane of the cochlea. 4 Immunohistochemical method: Oxidation was detected by immunohistochemical method Stress product 4-HNE was used to evaluate the level of oxidative stress. 5 TUNEL apoptosis detection: TUNEL apoptosis method was used to observe the apoptosis. 6 Western blot: Western blot was used to detect the expression of Cx26 and CX30 in each group. Results: 1 General observation: The blank control group mice were normal. The ears of the mice given methionine before noise grew normally, and the ears of the mice given methionine before noise grew worse than that of the control group. The ears of the mice given methionine after noise grew normally, and the ears of the mice given methionine after noise grew worse than that of the control group. (abr) test results: there was no significant difference in ABR threshold between the groups before the experiment (p0.05). the ABR threshold of the blank control group was (16.25 + 3.58) dbspl. the ABR threshold of the noise group was (16.00 + 3.37) dbspl. the ABR threshold of the methionine group was (16.00 + 2.69) dbspl before the noise. the ABR threshold of the methionine group was (15.75) dbspl after the noise. Immediately after noise exposure: the ABR threshold of the control group was (16.75 (3.74) dbspl, the noise group was (58.75 (6.80) dbspl, the methionine group was (52.75 (9.01) dbspl before noise exposure, and the methionine group was (60.00 (5.14) dbspl after noise exposure, except the blank control group. There were significant differences in the self-matching of the three groups of mice before and after noise exposure (p0.05); the ABR threshold of the control group was (16.50 (+ 3.16) dbspl on the fourth day after noise exposure; the ABR threshold of the noise group was (36.25 (+ 7.48) dbspl; the ABR threshold of the methionine group was (23.00 (+ 3.29) dbspl) before noise exposure; and the control group was (16.50 (+ 3.16) dbspl) after noise exposure. The ABR threshold of mice in the methionine group was (23.25 (+ 3.13) dbspl. Except for the blank control group, the other three groups of mice were compared in pairs. There was significant difference between the noise group and the methionine group before and after the noise treatment (p0.05). There was no significant difference between the methionine group before and after the noise treatment (p0.05). 3 basement membrane coating. He staining and myosin-VI staining results: in the blank control group, the outer hair cells were well-distributed, the outline of cells was clear, no degeneration and other pathological changes were found. Compared with the blank control group, the cochlear basement membrane hair cells also showed scattered deletions, mainly in the outer hair cells, and the inner hair cells were more intact. 44-hne immunohistochemical staining results: blank control group The expression of 4-HNE was not found in the basement membrane, stria vascularis and helical ligament in the cochlear tissue slices of mice; the expression of 4-HNE was more obvious in the basement membrane, stria vascularis and helical ligament of the cochlea in the noise group; the expression of 4-HNE was found in the basement membrane, stria vascularis and helical ligament in the methionine group before noise and in the methionine group after noise. But the results of TUNEL apoptosis were weaker than those of noise group. 5 TUNEL apoptosis test showed that no apoptotic cells were found in the basilar membrane, stria vascularis and spiral ligament of the cochlea in the blank control group, but in the noise group, apoptotic cells were found in the above three structures, and the expression was obvious. The results of Cx26 and Cx30 Western blot showed that the expression of Cx26 and Cx30 in the noise group was significantly weaker than that in the blank control group. The expression of Cx26 and Cx30 in the methionine group before noise and in the methionine group after noise were stronger than that in the blank control group before noise. There was no significant difference in the expression of Cx26 and Cx30 between the methionine group and the methionine group. Conclusion: 1. Noise deafness mainly damages the inner and outer hair cells of the cochlear Corti organ, and mainly damages the bottom to outer hair cells. Methionine causes hearing loss in noise deafness. Noise can induce noise-induced deafness through reactive oxygen species and cell apoptosis, while methionine has a protective effect on reactive oxygen species and cell apoptosis. 3 Connexin Cx26 and Cx30 may be related to the pathogenesis of noise-induced deafness, and methionine can protect Cx2. The results showed that methionine could prevent and treat noise-induced deafness.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R764.43

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王博;胡博華;楊仕明;;耳蝸內(nèi)細(xì)胞連接蛋白研究進(jìn)展[J];中華耳科學(xué)雜志;2015年01期

2 戴清蕾;薛希均;嚴(yán)旭坤;戴樸;;噪聲性耳聾相關(guān)的細(xì)胞病理生理變化概述[J];中華耳鼻咽喉頭頸外科雜志;2014年07期

3 葛振民;馬樞;賈曉青;;甲硫氨酸對噪聲性聽力損失的預(yù)防作用研究[J];聽力學(xué)及言語疾病雜志;2014年06期

4 葛薩薩;鄭貴亮;周義德;;氧化應(yīng)激在噪聲性聾發(fā)病中的作用機(jī)制及干預(yù)[J];中華耳科學(xué)雜志;2014年02期

5 陳立偉;于寧;翟所強(qiáng);;氧化應(yīng)激反應(yīng)與噪聲性耳聾[J];中華耳科學(xué)雜志;2014年01期

6 萬甜甜;王璐;倪月秋;;噪聲性耳聾的研究進(jìn)展[J];沈陽醫(yī)學(xué)院學(xué)報;2014年01期

7 韓維舉;陳星睿;;噪聲暴露引起耳蝸損傷機(jī)制的研究[J];中華耳科學(xué)雜志;2013年03期

8 張璞;林曦;曲雁;;Connexin蛋白與耳聾[J];中華耳科學(xué)雜志;2013年01期

9 王亞菲;王潔;朱慶春;邱建華;;噪聲對小鼠耳蝸外側(cè)壁縫隙連接蛋白Connexin26表達(dá)的影響[J];聽力學(xué)及言語疾病雜志;2013年03期

10 王亞菲;王娟;張鵬志;米文娟;蔣興旺;王潔;邱建華;;噪聲對小鼠耳蝸外側(cè)壁縫隙連接蛋白Connexin31表達(dá)的影響[J];聽力學(xué)及言語疾病雜志;2012年05期

，

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