長期噪聲暴露對大鼠海馬、皮層CRF信號系統(tǒng)的影響及其與p-tau關(guān)系的研究
發(fā)布時間：2018-09-12 17:29
【摘要】：隨著現(xiàn)代工業(yè)社會的不斷發(fā)展,噪聲危害日益嚴(yán)重,交通、社會生活、工業(yè)及一些特殊環(huán)境噪聲無時無刻不在影響人們的工作、學(xué)習(xí)和生活。前期研究結(jié)果顯示,長時間慢性噪聲暴露可以引發(fā)大鼠海馬、皮層部位的tau蛋白過度磷酸化及Aβ異常蓄積等阿爾茨海默病樣改變(Alzheimer’s disease,AD),但噪聲刺激致神經(jīng)系統(tǒng)損傷的具體機(jī)制尚不清楚。在非噪聲應(yīng)激導(dǎo)致的tau異常磷酸化過程中,促腎上腺皮質(zhì)激素釋放激素(Corticotropin-releasing factor,C RF)信號系統(tǒng)起到了關(guān)鍵的調(diào)節(jié)作用。本課題旨在以CRF信號系統(tǒng)為研究切入點,擬從基因、蛋白及定位表達(dá)三個層面探討噪聲暴露對海馬、皮層CRF信號系統(tǒng)的影響及其與p-tau的定位關(guān)系,為進(jìn)一步認(rèn)識噪聲暴露致AD樣神經(jīng)系統(tǒng)損傷效應(yīng)機(jī)制及有效預(yù)防措施的制定提供理論依據(jù)�！狙芯磕康摹刻接懠懊鞔_長期噪聲暴露后大鼠HPA軸及海馬、皮層CRF信號系統(tǒng)的變化及其與AD樣變之間的作用關(guān)系,以期進(jìn)一步認(rèn)識長期噪聲暴露致AD樣變的可能的信號機(jī)制�！狙芯糠椒ā繉嶒瀸ο筮x擇Wistar健康雄性大鼠(200-220g)64只,在適應(yīng)實驗環(huán)境5天后,隨機(jī)分為對照組和噪聲暴露組,每組32只,暴露條件為95 dB白噪聲,4 h/d×30 d,停止噪聲暴露分別恢復(fù)0、3、7、14 d,對照組動物除不接受噪聲暴露外,其他條件均與暴露組相同。兩組實驗動物分別在各個恢復(fù)期(0、3、7、14 d)相同時間點處死大鼠,每組取6只大鼠通過腹主動脈取血分離血漿,采用Elisa法測定血漿皮質(zhì)酮(CORT)水平;分離大鼠海馬、皮層組織分別采用RT-PCR法檢測CRF及其受體CRFR1、CRFR2mRNA的表達(dá)變化,Western blot法檢測CRF、CRFR1、CRFR2的蛋白表達(dá)變化。每組剩余兩只大鼠采用水合氯醛麻醉后先后用生理鹽水和4%的多聚甲醛進(jìn)行心臟灌注,灌注結(jié)束后分離出大鼠大腦組織后進(jìn)行一系列脫水、石蠟包埋,采用石蠟切片機(jī)切取6um厚的海馬、皮層組織切片。采用免疫熒光雙標(biāo)法對組織切片進(jìn)行CRF與p-tau的雙重標(biāo)記,在熒光顯微鏡下觀察海馬、皮層神經(jīng)元中CRF與p-tau的表達(dá)定位情況�！狙芯拷Y(jié)果】1.長期噪聲暴露對大鼠血漿皮質(zhì)酮的影響停止噪聲接觸后分別恢復(fù)0、3、7、14天時對噪聲接觸組和對照組大鼠血漿皮質(zhì)酮表達(dá)水平進(jìn)行Elisa檢測,實驗結(jié)果顯示從停止噪聲接觸后恢復(fù)0天時開始噪聲接觸組大鼠血漿皮質(zhì)酮水平顯著高于對照組,這一趨勢一直持續(xù)到恢復(fù)7天之后,差異有統(tǒng)計學(xué)意義(P0.05),到恢復(fù)14天時噪聲接觸組大鼠血漿皮質(zhì)酮恢復(fù)到對照組水平,差異無統(tǒng)計學(xué)意義(P0.05)。2.長期噪聲暴露對大鼠海馬、皮層CRF及其受體CRFR1、CRFR2基因、蛋白表達(dá)的影響停止噪聲刺激接觸后分別恢復(fù)0、3、7、14天時對噪聲暴露組和對照組大鼠海馬、皮層CRF、CRFR1、CRFR2 mRNA和蛋白表達(dá)水平進(jìn)行檢測,實驗結(jié)果顯示,在恢復(fù)0天時噪聲接觸組大鼠CRF和CRFR1 mRNA水平顯著高于對照組,且這一顯著趨勢一直持續(xù)到恢復(fù)7天之后,差異有顯著性(P0.05),到第14天時恢復(fù)到正常水平;兩者蛋白表達(dá)水平在各個時間點的變化情況同其基因表達(dá)趨勢相同,同樣在第14天時恢復(fù)到正常水平;CRFR2 mRNA水平在停止噪聲接觸恢復(fù)0天時噪聲接觸組與對照組無顯著性差異,到恢復(fù)3天時噪聲接觸組表達(dá)水平顯著升高,這一趨勢一直持續(xù)到恢復(fù)第14天之后(P0.05);其蛋白表達(dá)水平在各個時間點的變化情況同其基因表達(dá)變化趨勢相同。提示噪聲刺激接觸顯著影響了大鼠海馬、皮層CRF系統(tǒng)的表達(dá)情況。3.大鼠海馬、皮層神經(jīng)元CRF與p-tau表達(dá)定位情況檢測在大鼠海馬和皮層組織中都能檢測到CRF和p-tau的表達(dá),為進(jìn)一步檢測兩者的定位情況采用免疫熒光雙標(biāo)法對海馬和皮層組織切片進(jìn)行熒光標(biāo)記,標(biāo)記結(jié)果顯示,CRF(綠色熒光標(biāo)記)在神經(jīng)元胞漿中存在表達(dá),p-tau(紅色熒光標(biāo)記)也定位于神經(jīng)元胞漿中,將兩者重疊后Merge(黃色)發(fā)現(xiàn)在部分神經(jīng)元胞漿內(nèi)CRF與p-tau存在同時表達(dá),結(jié)果提示在海馬、皮層組織神經(jīng)元中CRF與p-tau存在共定位現(xiàn)象�！狙芯拷Y(jié)論】1.長期噪聲暴露能夠刺激機(jī)體啟動HPA軸,引起大鼠血漿皮質(zhì)酮水平的持續(xù)性顯著升高,使機(jī)體對應(yīng)激刺激做出反應(yīng)。2.長期噪聲暴露能夠顯著影響大鼠海馬、皮層CRF信號系統(tǒng)的表達(dá)情況,且具體的表現(xiàn)及機(jī)制存在差異;CRFR2基因及蛋白呈現(xiàn)高表達(dá)現(xiàn)象。3.免疫熒光雙標(biāo)法海馬、皮層神經(jīng)元中CRF與p-tau存在共定位表達(dá)現(xiàn)象,進(jìn)一步加強了長期噪聲暴露中CRF信號系統(tǒng)參與tau異常磷酸化進(jìn)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.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R12
本文編號：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.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R12
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 張偉文;;現(xiàn)代城市噪聲污染分析及治理措施建議[J];企業(yè)科技與發(fā)展;2010年12期
相關(guān)博士學(xué)位論文 前1條
1 崔博;噪聲暴露對認(rèn)知功能的影響及其海馬機(jī)制研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2010年
，本文編號：2239735
本文鏈接：http://sikaile.net/shengtaihuanjingbaohulunwen/2239735.html
最近更新
教材專著
