噪声刺激前后Mn-SOD在大鼠螺旋神经节表达与活性的区域性差异
发布时间：2018-12-15 10:17
【摘要】：1目的 感音神经性聋是耳科常见的疾病，高频区听力易损性是其最显著的特点。有研究显示，感音神经性聋的发生与耳蜗感觉神经细胞的氧化损伤存在着密切的关系。而螺旋神经节细胞作为耳蜗内重要的感觉神经细胞虽然在感音神经性聋发生的早期往往没有明显的形态学损伤，但其同样受到了氧化应激作用的影响，致使氧化应激相关分子的表达与性质发生了变化。且这种变化在螺旋神经节的不同区域之间存在着差异。其中抗氧化剂的区域性分布差异及活性变化体现了不同区域的细胞抗氧化能力的差异。本研究中，我们主要验证了在噪声刺激前后线粒体内抗氧化酶锰超氧化物歧化酶（Mn-SOD）在大鼠螺旋神经节不同区域表达和活性的差异与高频听力易损性在区域上的相关性。 2方法 本实验通过给予白噪声115dB2h连续3d，建立SD大鼠噪声性聋的动物模型，ABR测听分析不同频率的噪声损伤情况;免疫组织化学染色法和western-blot分析噪声暴露前后Mn-SOD在螺旋神经节不同区域的表达水平;黄嘌呤氧化酶法检测Mn-SOD在螺旋神经节不同区域的活性及变化趋势；荧光实时定量PCR技术来测定噪声刺激前后Mn-SOD的mRNA在螺旋神经节不同区域的表达差异。 3结果 ABR测听结果显示：大鼠噪声暴露后第1、3、14d与暴露前1d相比，4、8、16、20、32kHz的ABR阈值均明显上移，而且以高频区听阈变化更为显著；免疫组织化学染色及western-blot结果显示：在正常对照组中,顶部螺旋神经节的Mn-SOD的表达较底部明显；而噪声暴露后，Mn-SOD在螺旋神经节的表达较对照组相应部位均显著增高；且顶部较底部的表达仍然更为显著；黄嘌呤氧化酶法Mn-SOD活性测定结果显示：在正常情况下,顶部螺旋神经节的Mn-SOD活性与底部相比无统计学差异，，而在噪声暴露后，Mn-SOD的活性则较对照组相应部位均有降低，而且底部较顶部的变化更为显著；荧光实时定量PCR结果显示：在正常情况下,顶部螺旋神经节的Mn-SODmRNA的表达较底部明显；而噪声暴露后，Mn-SODmRNA在螺旋神经节的表达较对照组相应部位均显著增高；且顶部仍较底部的表达量更大。 4结论 以往研究表明，在噪声性耳聋的早期，以底回外毛细胞的损伤为主，而螺旋神经节则没有发生明显的形态学改变。然而，我们的研究表明，噪声刺激同样在早期既可引起对螺旋神经节细胞的氧化应激作用，并导致螺旋神经节细胞内相关的抗氧化酶类表达水平发生了改变。而且表现为顶部螺旋神经节细胞的抗氧化能力显著高于底部。抗氧化能力的区域性差异与噪声性耳聋的高频听力易损性的特点，以及其对应的毛细胞损伤区域相一致。这一现象，为今后进一步研究噪声性耳以及聋感音神经性聋的氧化损伤机制及高频听力易损性的影响因素提供了新的思路。
[Abstract]:Objective sensorineural deafness is a common disease in otology. Studies have shown that the occurrence of sensorineural deafness is closely related to oxidative injury of sensory neurons in the cochlea. As an important sensory nerve cell in the cochlea, the spiral ganglion cells had no obvious morphological damage in the early stage of sensorineural hearing loss, but they were also affected by oxidative stress. As a result, the expression and properties of oxidative stress related molecules changed. The changes were different in different regions of the spiral ganglion. The regional distribution of antioxidants and the changes of their activities reflect the difference of cell antioxidant capacity in different regions. In this study, we examined the correlation between the expression and activity of antioxidant enzyme manganese superoxide dismutase (Mn-SOD) in mitochondria in different regions of rat spiral ganglion before and after noise stimulation and the high frequency hearing vulnerability. Methods the animal model of noise induced deafness in SD rats was established by giving white noise 115dB2h for 3 consecutive days. ABR audiometry was used to analyze the noise damage at different frequencies. Immunohistochemical staining and western-blot were used to analyze the expression level of Mn-SOD in different regions of spiral ganglion before and after noise exposure, and xanthine oxidase method was used to detect the activity and change trend of Mn-SOD in different regions of spiral ganglion. Fluorescence real-time quantitative PCR was used to detect the difference of Mn-SOD mRNA expression in different regions of spiral ganglion before and after noise stimulation. Results the results of ABR audiometry showed that compared with the first day after noise exposure, the ABR thresholds of the rats at the 1st day after noise exposure were significantly higher than those at the first day before exposure, and the changes of the ABR thresholds in the high frequency region were more significant than those in the first day after noise exposure. Immunohistochemical staining and western-blot results showed that the expression of Mn-SOD in the parietal spiral ganglion was significantly higher than that in the bottom of the control group. After noise exposure, the expression of Mn-SOD in the spiral ganglion was significantly higher than that in the control group, and the expression of Mn-SOD was still more significant at the top than at the bottom. The results of Mn-SOD activity determination by xanthine oxidase method showed that the Mn-SOD activity of the parietal spiral ganglion was not significantly different from that of the bottom under normal conditions, but after noise exposure, there was no significant difference in the activity of Mn-SOD in the parietal spiral ganglion. The activity of Mn-SOD was lower than that of the control group, and the change of the bottom was more significant than that of the top. The results of real-time quantitative PCR showed that the expression of Mn-SODmRNA in the parietal spiral ganglion was significantly higher than that in the bottom, and the expression of Mn-SODmRNA in the spiral ganglion was significantly higher than that in the control group after noise exposure. And the expression at the top is still larger than that at the bottom. Conclusion in the early stage of noise-induced deafness, the damage of outer hair cells in the basal gyrus is the main one, while the spiral ganglion has no obvious morphologic changes in the early stage of noise-induced deafness. However, our studies have shown that noise stimulation can also cause oxidative stress in spiral ganglion cells and change the expression of related antioxidant enzymes in spiral ganglion cells. Moreover, the antioxidation ability of the parietal spiral ganglion cells was significantly higher than that of the bottom. The regional differences in antioxidant capacity are consistent with the characteristics of high frequency hearing vulnerability and the corresponding hair cell damage area of noise-induced deafness. This phenomenon provides a new idea for the further study of oxidative damage mechanism of noise-induced hearing loss and deaf-sensorineural hearing loss and the influencing factors of high-frequency hearing vulnerability.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R764.431
本文编号：2380469
[Abstract]:Objective sensorineural deafness is a common disease in otology. Studies have shown that the occurrence of sensorineural deafness is closely related to oxidative injury of sensory neurons in the cochlea. As an important sensory nerve cell in the cochlea, the spiral ganglion cells had no obvious morphological damage in the early stage of sensorineural hearing loss, but they were also affected by oxidative stress. As a result, the expression and properties of oxidative stress related molecules changed. The changes were different in different regions of the spiral ganglion. The regional distribution of antioxidants and the changes of their activities reflect the difference of cell antioxidant capacity in different regions. In this study, we examined the correlation between the expression and activity of antioxidant enzyme manganese superoxide dismutase (Mn-SOD) in mitochondria in different regions of rat spiral ganglion before and after noise stimulation and the high frequency hearing vulnerability. Methods the animal model of noise induced deafness in SD rats was established by giving white noise 115dB2h for 3 consecutive days. ABR audiometry was used to analyze the noise damage at different frequencies. Immunohistochemical staining and western-blot were used to analyze the expression level of Mn-SOD in different regions of spiral ganglion before and after noise exposure, and xanthine oxidase method was used to detect the activity and change trend of Mn-SOD in different regions of spiral ganglion. Fluorescence real-time quantitative PCR was used to detect the difference of Mn-SOD mRNA expression in different regions of spiral ganglion before and after noise stimulation. Results the results of ABR audiometry showed that compared with the first day after noise exposure, the ABR thresholds of the rats at the 1st day after noise exposure were significantly higher than those at the first day before exposure, and the changes of the ABR thresholds in the high frequency region were more significant than those in the first day after noise exposure. Immunohistochemical staining and western-blot results showed that the expression of Mn-SOD in the parietal spiral ganglion was significantly higher than that in the bottom of the control group. After noise exposure, the expression of Mn-SOD in the spiral ganglion was significantly higher than that in the control group, and the expression of Mn-SOD was still more significant at the top than at the bottom. The results of Mn-SOD activity determination by xanthine oxidase method showed that the Mn-SOD activity of the parietal spiral ganglion was not significantly different from that of the bottom under normal conditions, but after noise exposure, there was no significant difference in the activity of Mn-SOD in the parietal spiral ganglion. The activity of Mn-SOD was lower than that of the control group, and the change of the bottom was more significant than that of the top. The results of real-time quantitative PCR showed that the expression of Mn-SODmRNA in the parietal spiral ganglion was significantly higher than that in the bottom, and the expression of Mn-SODmRNA in the spiral ganglion was significantly higher than that in the control group after noise exposure. And the expression at the top is still larger than that at the bottom. Conclusion in the early stage of noise-induced deafness, the damage of outer hair cells in the basal gyrus is the main one, while the spiral ganglion has no obvious morphologic changes in the early stage of noise-induced deafness. However, our studies have shown that noise stimulation can also cause oxidative stress in spiral ganglion cells and change the expression of related antioxidant enzymes in spiral ganglion cells. Moreover, the antioxidation ability of the parietal spiral ganglion cells was significantly higher than that of the bottom. The regional differences in antioxidant capacity are consistent with the characteristics of high frequency hearing vulnerability and the corresponding hair cell damage area of noise-induced deafness. This phenomenon provides a new idea for the further study of oxidative damage mechanism of noise-induced hearing loss and deaf-sensorineural hearing loss and the influencing factors of high-frequency hearing vulnerability.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R764.431
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