噪聲刺激前后Mn-SOD在大鼠螺旋神經(jīng)節(jié)表達與活性的區(qū)域性差異
發(fā)布時間：2018-12-15 10:17
【摘要】：1目的 感音神經(jīng)性聾是耳科常見的疾病，高頻區(qū)聽力易損性是其最顯著的特點。有研究顯示，感音神經(jīng)性聾的發(fā)生與耳蝸感覺神經(jīng)細胞的氧化損傷存在著密切的關(guān)系。而螺旋神經(jīng)節(jié)細胞作為耳蝸內(nèi)重要的感覺神經(jīng)細胞雖然在感音神經(jīng)性聾發(fā)生的早期往往沒有明顯的形態(tài)學(xué)損傷，但其同樣受到了氧化應(yīng)激作用的影響，致使氧化應(yīng)激相關(guān)分子的表達與性質(zhì)發(fā)生了變化。且這種變化在螺旋神經(jīng)節(jié)的不同區(qū)域之間存在著差異。其中抗氧化劑的區(qū)域性分布差異及活性變化體現(xiàn)了不同區(qū)域的細胞抗氧化能力的差異。本研究中，我們主要驗證了在噪聲刺激前后線粒體內(nèi)抗氧化酶錳超氧化物歧化酶（Mn-SOD）在大鼠螺旋神經(jīng)節(jié)不同區(qū)域表達和活性的差異與高頻聽力易損性在區(qū)域上的相關(guān)性。 2方法 本實驗通過給予白噪聲115dB2h連續(xù)3d，建立SD大鼠噪聲性聾的動物模型，ABR測聽分析不同頻率的噪聲損傷情況;免疫組織化學(xué)染色法和western-blot分析噪聲暴露前后Mn-SOD在螺旋神經(jīng)節(jié)不同區(qū)域的表達水平;黃嘌呤氧化酶法檢測Mn-SOD在螺旋神經(jīng)節(jié)不同區(qū)域的活性及變化趨勢；熒光實時定量PCR技術(shù)來測定噪聲刺激前后Mn-SOD的mRNA在螺旋神經(jīng)節(jié)不同區(qū)域的表達差異。 3結(jié)果 ABR測聽結(jié)果顯示：大鼠噪聲暴露后第1、3、14d與暴露前1d相比，4、8、16、20、32kHz的ABR閾值均明顯上移，而且以高頻區(qū)聽閾變化更為顯著；免疫組織化學(xué)染色及western-blot結(jié)果顯示：在正常對照組中,頂部螺旋神經(jīng)節(jié)的Mn-SOD的表達較底部明顯；而噪聲暴露后，Mn-SOD在螺旋神經(jīng)節(jié)的表達較對照組相應(yīng)部位均顯著增高；且頂部較底部的表達仍然更為顯著；黃嘌呤氧化酶法Mn-SOD活性測定結(jié)果顯示：在正常情況下,頂部螺旋神經(jīng)節(jié)的Mn-SOD活性與底部相比無統(tǒng)計學(xué)差異，，而在噪聲暴露后，Mn-SOD的活性則較對照組相應(yīng)部位均有降低，而且底部較頂部的變化更為顯著；熒光實時定量PCR結(jié)果顯示：在正常情況下,頂部螺旋神經(jīng)節(jié)的Mn-SODmRNA的表達較底部明顯；而噪聲暴露后，Mn-SODmRNA在螺旋神經(jīng)節(jié)的表達較對照組相應(yīng)部位均顯著增高；且頂部仍較底部的表達量更大。 4結(jié)論 以往研究表明，在噪聲性耳聾的早期，以底回外毛細胞的損傷為主，而螺旋神經(jīng)節(jié)則沒有發(fā)生明顯的形態(tài)學(xué)改變。然而，我們的研究表明，噪聲刺激同樣在早期既可引起對螺旋神經(jīng)節(jié)細胞的氧化應(yīng)激作用，并導(dǎo)致螺旋神經(jīng)節(jié)細胞內(nèi)相關(guān)的抗氧化酶類表達水平發(fā)生了改變。而且表現(xiàn)為頂部螺旋神經(jīng)節(jié)細胞的抗氧化能力顯著高于底部。抗氧化能力的區(qū)域性差異與噪聲性耳聾的高頻聽力易損性的特點，以及其對應(yīng)的毛細胞損傷區(qū)域相一致。這一現(xiàn)象，為今后進一步研究噪聲性耳以及聾感音神經(jīng)性聾的氧化損傷機制及高頻聽力易損性的影響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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R764.431
本文編號：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R764.431
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