长期噪声暴露对大鼠海马、皮层CRF信号系统的影响及其与p-tau关系的研究
发布时间：2018-09-12 17:29
【摘要】：随着现代工业社会的不断发展,噪声危害日益严重,交通、社会生活、工业及一些特殊环境噪声无时无刻不在影响人们的工作、学习和生活。前期研究结果显示,长时间慢性噪声暴露可以引发大鼠海马、皮层部位的tau蛋白过度磷酸化及Aβ异常蓄积等阿尔茨海默病样改变(Alzheimer’s disease,AD),但噪声刺激致神经系统损伤的具体机制尚不清楚。在非噪声应激导致的tau异常磷酸化过程中,促肾上腺皮质激素释放激素(Corticotropin-releasing factor,C RF)信号系统起到了关键的调节作用。本课题旨在以CRF信号系统为研究切入点,拟从基因、蛋白及定位表达三个层面探讨噪声暴露对海马、皮层CRF信号系统的影响及其与p-tau的定位关系,为进一步认识噪声暴露致AD样神经系统损伤效应机制及有效预防措施的制定提供理论依据。【研究目的】探讨及明确长期噪声暴露后大鼠HPA轴及海马、皮层CRF信号系统的变化及其与AD样变之间的作用关系,以期进一步认识长期噪声暴露致AD样变的可能的信号机制。【研究方法】实验对象选择Wistar健康雄性大鼠(200-220g)64只,在适应实验环境5天后,随机分为对照组和噪声暴露组,每组32只,暴露条件为95 dB白噪声,4 h/d×30 d,停止噪声暴露分别恢复0、3、7、14 d,对照组动物除不接受噪声暴露外,其他条件均与暴露组相同。两组实验动物分别在各个恢复期(0、3、7、14 d)相同时间点处死大鼠,每组取6只大鼠通过腹主动脉取血分离血浆,采用Elisa法测定血浆皮质酮(CORT)水平;分离大鼠海马、皮层组织分别采用RT-PCR法检测CRF及其受体CRFR1、CRFR2mRNA的表达变化,Western blot法检测CRF、CRFR1、CRFR2的蛋白表达变化。每组剩余两只大鼠采用水合氯醛麻醉后先后用生理盐水和4%的多聚甲醛进行心脏灌注,灌注结束后分离出大鼠大脑组织后进行一系列脱水、石蜡包埋,采用石蜡切片机切取6um厚的海马、皮层组织切片。采用免疫荧光双标法对组织切片进行CRF与p-tau的双重标记,在荧光显微镜下观察海马、皮层神经元中CRF与p-tau的表达定位情况。【研究结果】1.长期噪声暴露对大鼠血浆皮质酮的影响停止噪声接触后分别恢复0、3、7、14天时对噪声接触组和对照组大鼠血浆皮质酮表达水平进行Elisa检测,实验结果显示从停止噪声接触后恢复0天时开始噪声接触组大鼠血浆皮质酮水平显著高于对照组,这一趋势一直持续到恢复7天之后,差异有统计学意义(P0.05),到恢复14天时噪声接触组大鼠血浆皮质酮恢复到对照组水平,差异无统计学意义(P0.05)。2.长期噪声暴露对大鼠海马、皮层CRF及其受体CRFR1、CRFR2基因、蛋白表达的影响停止噪声刺激接触后分别恢复0、3、7、14天时对噪声暴露组和对照组大鼠海马、皮层CRF、CRFR1、CRFR2 mRNA和蛋白表达水平进行检测,实验结果显示,在恢复0天时噪声接触组大鼠CRF和CRFR1 mRNA水平显著高于对照组,且这一显著趋势一直持续到恢复7天之后,差异有显著性(P0.05),到第14天时恢复到正常水平;两者蛋白表达水平在各个时间点的变化情况同其基因表达趋势相同,同样在第14天时恢复到正常水平;CRFR2 mRNA水平在停止噪声接触恢复0天时噪声接触组与对照组无显著性差异,到恢复3天时噪声接触组表达水平显著升高,这一趋势一直持续到恢复第14天之后(P0.05);其蛋白表达水平在各个时间点的变化情况同其基因表达变化趋势相同。提示噪声刺激接触显著影响了大鼠海马、皮层CRF系统的表达情况。3.大鼠海马、皮层神经元CRF与p-tau表达定位情况检测在大鼠海马和皮层组织中都能检测到CRF和p-tau的表达,为进一步检测两者的定位情况采用免疫荧光双标法对海马和皮层组织切片进行荧光标记,标记结果显示,CRF(绿色荧光标记)在神经元胞浆中存在表达,p-tau(红色荧光标记)也定位于神经元胞浆中,将两者重叠后Merge(黄色)发现在部分神经元胞浆内CRF与p-tau存在同时表达,结果提示在海马、皮层组织神经元中CRF与p-tau存在共定位现象。【研究结论】1.长期噪声暴露能够刺激机体启动HPA轴,引起大鼠血浆皮质酮水平的持续性显著升高,使机体对应激刺激做出反应。2.长期噪声暴露能够显著影响大鼠海马、皮层CRF信号系统的表达情况,且具体的表现及机制存在差异;CRFR2基因及蛋白呈现高表达现象。3.免疫荧光双标法海马、皮层神经元中CRF与p-tau存在共定位表达现象,进一步加强了长期噪声暴露中CRF信号系统参与tau异常磷酸化进程的可能性。
[Abstract]:With the continuous development of modern industrial society, noise hazards become increasingly serious. Traffic, social life, industry and some special environmental noises all the time affect people's work, study and life. Alzheimer's disease (AD), such as chronic accumulation, is not well understood. Corticotropin-releasing factor (C RF) signaling system plays a key role in the abnormal phosphorylation of tau induced by non-noise stress. The purpose of this study is to explore the effects of noise exposure on the CRF signaling system in the hippocampus and cortex and its relationship with the localization of p-tau from three aspects of gene, protein and localization expression, so as to provide a theoretical basis for further understanding the mechanism of AD-like nervous system damage induced by noise exposure and formulating effective preventive measures. [Objective] To explore and clarify the changes of CRF signaling system in HPA axis, hippocampus and cortex of rats after long-term noise exposure and the relationship between CRF signaling system and AD-like changes, so as to further understand the possible signal mechanism of AD-like changes induced by long-term noise exposure. [Methods] Wistar healthy male rats (200-220g) were selected as subjects. Sixty-four rats were randomly divided into control group and noise exposure group after 5 days of adaptation to the experimental environment. The exposure conditions were 95 dB white noise, 4 h/d *30 d, and the stopping noise exposure was restored to 0,3,7,14 d, respectively. The other conditions of the control group were the same as those of the exposure group except that they did not receive noise exposure. D) At the same time point, rats were sacrificed, 6 rats in each group were taken blood from abdominal aorta to isolate plasma, and the levels of plasma corticosterone (CORT) were measured by Elisa method; the expression of CRF, CRFR1 and CRFR2 mRNA in hippocampus and cortex of rats were detected by RT-PCR, and the expression of CRF, CRFR1 and CRFR2 protein was detected by Western blot. The remaining two rats in each group were anesthetized with chloral hydrate and perfused with normal saline and 4% paraformaldehyde. After perfusion, the brain tissues of rats were separated and dehydrated. Paraffin embedded, 6-um-thick hippocampus and cortical tissue were cut by paraffin section machine. Immunofluorescence double labeling method was used for tissue section. The expression and localization of CRF and p-tau in hippocampal and cortical neurons were observed under fluorescence microscope with double labeling of CRF and p-tau. The results of Elisa test showed that the level of plasma corticosterone in the noise exposure group was significantly higher than that in the control group from 0 days after the noise exposure was stopped, and the trend continued until 7 days after the noise exposure (P 0.05). There was no significant difference (P 0.05). 2. The expression of CRF, CRFR1 and CRFR2 mRNA and protein in hippocampus and cortex of rats exposed to noise and control group were detected at 0, 3, 7 and 14 days after noise exposure, respectively. The levels of CRF and CRFR1 mRNA in the noise exposed group were significantly higher than those in the control group at day 0, and the difference was significant (P 0.05) after 7 days of recovery, and returned to normal level at day 14. The expression of CRF and CRFR1 mRNA in the noise exposed group was the same as that in the control group at day 14. CRFR2 mRNA level returned to normal level at day 1; CRFR2 mRNA level had no significant difference between the noise exposure group and the control group at day 0, but increased significantly at day 3, and this trend continued until day 14 (P 0.05); the change of CRFR2 protein expression level at each time point was the same as its base. The expression of CRF and p-tau in hippocampus and cortex of rats were detected by immunofluorescence assay. The localization of CRF and p-tau in hippocampus and cortex of rats was detected by immunofluorescence assay. The results showed that CRF (green fluorescent marker) was expressed in the cytoplasm of neurons and p-tau (red fluorescent marker) was also localized in the cytoplasm of neurons. Merge (yellow) found that CRF and p-tau were simultaneously expressed in the cytoplasm of some neurons after overlapping the two markers. In the hippocampus, there is co-localization between CRF and p-tau in cortical neurons. [Conclusion] 1. Long-term noise exposure can stimulate the body to initiate the HPA axis, resulting in a significant increase in plasma corticosterone levels in rats, making the body respond to stress stimulation. 2. Long-term noise exposure can significantly affect the hippocampus, cortical CRF signal. The expression of CRFR2 gene and protein showed high expression. 3. The co-localization of CRF and p-tau in hippocampus and cortex neurons by immunofluorescence double labeling method further enhanced the possibility of CRF signaling system participating in the abnormal phosphorylation of tau during long-term noise exposure.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R12
本文编号：2239735
[Abstract]:With the continuous development of modern industrial society, noise hazards become increasingly serious. Traffic, social life, industry and some special environmental noises all the time affect people's work, study and life. Alzheimer's disease (AD), such as chronic accumulation, is not well understood. Corticotropin-releasing factor (C RF) signaling system plays a key role in the abnormal phosphorylation of tau induced by non-noise stress. The purpose of this study is to explore the effects of noise exposure on the CRF signaling system in the hippocampus and cortex and its relationship with the localization of p-tau from three aspects of gene, protein and localization expression, so as to provide a theoretical basis for further understanding the mechanism of AD-like nervous system damage induced by noise exposure and formulating effective preventive measures. [Objective] To explore and clarify the changes of CRF signaling system in HPA axis, hippocampus and cortex of rats after long-term noise exposure and the relationship between CRF signaling system and AD-like changes, so as to further understand the possible signal mechanism of AD-like changes induced by long-term noise exposure. [Methods] Wistar healthy male rats (200-220g) were selected as subjects. Sixty-four rats were randomly divided into control group and noise exposure group after 5 days of adaptation to the experimental environment. The exposure conditions were 95 dB white noise, 4 h/d *30 d, and the stopping noise exposure was restored to 0,3,7,14 d, respectively. The other conditions of the control group were the same as those of the exposure group except that they did not receive noise exposure. D) At the same time point, rats were sacrificed, 6 rats in each group were taken blood from abdominal aorta to isolate plasma, and the levels of plasma corticosterone (CORT) were measured by Elisa method; the expression of CRF, CRFR1 and CRFR2 mRNA in hippocampus and cortex of rats were detected by RT-PCR, and the expression of CRF, CRFR1 and CRFR2 protein was detected by Western blot. The remaining two rats in each group were anesthetized with chloral hydrate and perfused with normal saline and 4% paraformaldehyde. After perfusion, the brain tissues of rats were separated and dehydrated. Paraffin embedded, 6-um-thick hippocampus and cortical tissue were cut by paraffin section machine. Immunofluorescence double labeling method was used for tissue section. The expression and localization of CRF and p-tau in hippocampal and cortical neurons were observed under fluorescence microscope with double labeling of CRF and p-tau. The results of Elisa test showed that the level of plasma corticosterone in the noise exposure group was significantly higher than that in the control group from 0 days after the noise exposure was stopped, and the trend continued until 7 days after the noise exposure (P 0.05). There was no significant difference (P 0.05). 2. The expression of CRF, CRFR1 and CRFR2 mRNA and protein in hippocampus and cortex of rats exposed to noise and control group were detected at 0, 3, 7 and 14 days after noise exposure, respectively. The levels of CRF and CRFR1 mRNA in the noise exposed group were significantly higher than those in the control group at day 0, and the difference was significant (P 0.05) after 7 days of recovery, and returned to normal level at day 14. The expression of CRF and CRFR1 mRNA in the noise exposed group was the same as that in the control group at day 14. CRFR2 mRNA level returned to normal level at day 1; CRFR2 mRNA level had no significant difference between the noise exposure group and the control group at day 0, but increased significantly at day 3, and this trend continued until day 14 (P 0.05); the change of CRFR2 protein expression level at each time point was the same as its base. The expression of CRF and p-tau in hippocampus and cortex of rats were detected by immunofluorescence assay. The localization of CRF and p-tau in hippocampus and cortex of rats was detected by immunofluorescence assay. The results showed that CRF (green fluorescent marker) was expressed in the cytoplasm of neurons and p-tau (red fluorescent marker) was also localized in the cytoplasm of neurons. Merge (yellow) found that CRF and p-tau were simultaneously expressed in the cytoplasm of some neurons after overlapping the two markers. In the hippocampus, there is co-localization between CRF and p-tau in cortical neurons. [Conclusion] 1. Long-term noise exposure can stimulate the body to initiate the HPA axis, resulting in a significant increase in plasma corticosterone levels in rats, making the body respond to stress stimulation. 2. Long-term noise exposure can significantly affect the hippocampus, cortical CRF signal. The expression of CRFR2 gene and protein showed high expression. 3. The co-localization of CRF and p-tau in hippocampus and cortex neurons by immunofluorescence double labeling method further enhanced the possibility of CRF signaling system participating in the abnormal phosphorylation of tau during long-term noise exposure.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R12
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