蛋氨酸对噪声导致听觉损伤预防和治疗作用的研究
发布时间：2018-09-09 15:52
【摘要】：目的:研究蛋氨酸对噪声性耳聋小鼠听力损失的预防与治疗作用。通过动物模型来研究抗氧化剂蛋氨酸对实验对象耳蜗组织在功能学、形态学、氧化应激水平和凋亡水平以及连接蛋白26(Cx26)和连接蛋白30(Cx30)方面是否具有预防和治疗效果,并为研究噪声性耳聋的发病机制提供进一步依据。方法:1建立噪声性耳聋动物模型:选取健康40只雌性昆明小鼠,根据随机分组原则分为(1)空白对照组(n=10)(2)噪声组(n=10)(3)噪声前给予蛋氨酸组(n=10)(4)噪声后给予蛋氨酸组(n=10)。首先进行昆明小鼠外耳、中耳及内耳的检查,以排除影响内耳听力功能的因素。(1)空白对照组:不给予噪声处理,正常环境下生活,腹腔注射生理盐水400mg/Kg,2次/天,连续3天;(2)噪声组:噪声前3天给予生理盐水400mg/Kg腹腔注射,2次/天,连续3天,再给予100dB SPL白噪声,连续3天,每天持续8小时;(3)噪声前给予蛋氨酸组:噪声前3天给予蛋氨酸溶液400mg/Kg腹腔注射,连续3天,2次/天,再给予100dB SPL白噪声,连续3天,每天持续8小时;(4)噪声后给予蛋氨酸组:给予100dB SPL白噪声,连续3天,每天持续8小时,噪声3天结束后给予蛋氨酸溶液400mg/Kg腹腔注射,连续3天,2次/天。2监测噪声暴露前后小鼠听力功能变化:四组昆明小鼠在噪声暴露前、噪声暴露后即刻和噪声暴露后第4天,分别都接受听性脑干反应(ABR)来评估听力功能。3显微水平下耳蜗结构改变:通过耳蜗基底膜铺片HE染色并在普通光学显微镜下观察毛细胞损伤情况,再通过耳蜗基底膜铺片Myosin-Ⅵ染色在激光共聚焦显微镜下观察基底膜毛细胞损伤情况。4免疫组织化学方法:通过免疫组织化学方法,检测氧化应激产物4-羟基壬烯醛(4-HNE),以评价各组氧化应激水平。5 TUNEL凋亡检测:通过TUNEL凋亡方法,观察细胞凋亡情况。6westernblot法:使用westernblot法检测cx26和cx30两种缝隙链接蛋白在各组中的表达情况。结果:1一般观察结果:空白对照组小鼠正常生长,各项反应均好,耳廓对声音反应正常;噪声组小鼠不好动,耳廓对声音反应较迟钝;噪声前给予蛋氨酸组的小鼠正常生长,耳廓对声音反应较对照组差;噪声后给予蛋氨酸组的小鼠正常生长,耳廓对声音反应较对照组差。2听觉脑干反应(abr)测试结果:实验前各组动物abr阈值没有统计学差异(p0.05),空白对照组小鼠abr阈值为(16.25±3.58)dbspl,噪声组小鼠abr阈值为(16.00±3.37)dbspl,噪声前给予蛋氨酸组的小鼠abr阈值为(16.00±2.69)dbspl,噪声后给予蛋氨酸组的小鼠abr阈值为(15.75±2.90)dbspl;噪声暴露后即刻:空白对照组小鼠abr阈值为(16.75±3.74)dbspl,噪声组小鼠abr阈值为(58.75±6.80)dbspl,噪声前给予蛋氨酸组的小鼠abr阈值为(52.75±9.01)dbspl,噪声后给予蛋氨酸组的小鼠abr阈值为(60.00±5.14)dbspl,除空白对照组外,其余接受噪声处理的三组小鼠噪声暴露前后自身配对比较均有统计学差异(p0.05);噪声暴露后第4天,空白对照组小鼠abr阈值为(16.50±3.16)dbspl,噪声组小鼠abr阈值为(36.25±7.48)dbspl,噪声前给予蛋氨酸组的小鼠abr阈值为(23.00±3.29)dbspl,噪声后给予蛋氨酸组的小鼠abr阈值为(23.25±3.13)dbspl,除空白对照组外,其余三组小鼠两两比较,噪声组与噪声前、后给予蛋氨酸组间比较均有统计学差异(p0.05),而噪声前给予蛋氨酸组与噪声后给予蛋氨酸组之间比较无统计学差异(p0.05)。3基底膜铺片he染色和myosin-Ⅵ染色结果:空白对照组耳蜗内、外毛细胞分布整齐,细胞轮廓清晰,未发现变性等病理改变。噪声组与空白对照组相比,耳蜗基底膜毛细胞出现较为明显的缺失,底转外毛细胞尤为明显。噪声前给予蛋氨酸组与空白对照组相比,耳蜗基底膜毛细胞出现散在的缺失,可见变性等病理改变。噪声后给予蛋氨酸组与空白对照组相比,耳蜗基底膜毛细胞同样出现了散在的缺失,主要出现在外毛细胞,内毛细胞结构较为完整。44-hne免疫组织化学染色结果:空白对照组小鼠耳蜗组织切片中,在基底膜、血管纹和螺旋侧韧带中没有发现4-HNE的表达;噪声组耳蜗基底膜、血管纹和螺旋侧韧带中4-HNE表达较明显;噪声前给予蛋氨酸组和噪声后给予蛋氨酸组中,发现基底膜、血管纹和螺旋侧韧带中有4-HNE的表达,但均弱于噪声组。5 TUNEL凋亡检测结果:空白对照组实验动物耳蜗组织切片TUNEL染色中耳蜗基底膜、血管纹和螺旋侧韧带中没有发现凋亡细胞;而在噪声组上述三个结构中均发现凋亡细胞,并且表达明显;噪声前给予蛋氨酸组和噪声后给予蛋氨酸组均出现了凋亡细胞,但凋亡细胞数量明显少于噪声组。6 Cx26和Cx30 Western blot检测结果:噪声组中Cx26和Cx30的表达明显弱于空白对照组、噪声前给予蛋氨酸组和噪声后给予蛋氨酸组,而空白对照组中Cx26和Cx30的表达强于噪声前给予蛋氨酸组和噪声后给予蛋氨酸组,但噪声前给予蛋氨酸组和噪声后给予蛋氨酸组中Cx26和Cx30的表达没有明显差异。结论:1噪声性耳聋主要损伤耳蜗Corti器的内、外毛细胞,并且以底转外毛细胞损伤为主,蛋氨酸对噪声性耳聋造成的听力下降和形态结构破坏起到一定的保护作用。2噪声通过活性氧和细胞凋亡导致噪声性耳聋的出现,而蛋氨酸在活性氧和细胞凋亡水平上有一定的保护作用。3缝隙连接蛋白Cx26和Cx30可能与噪声性耳聋的致病机制相关,并且蛋氨酸可保护Cx26和Cx30在噪声下的表达。4噪声前给予蛋氨酸组和噪声后给予蛋氨酸组结果表明了蛋氨酸对噪声性耳聋有预防和治疗作用。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R764.43
本文编号：2232829
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R764.43
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