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学科主题: 耳鼻咽喉科学
题名:
模拟中长期航天失重和噪声环境对大鼠听觉系统影响的研究
作者: 陈娜
答辩日期: 2016-05-12
导师: 吴玮
专业: 耳鼻咽喉科学
授予单位: 北京大学
授予地点: 北京大学解放军306医院教学医院
学位: 硕士
关键词: 模拟失重 ; 中长期 ; 噪声 ; 听性脑干反应 ; 扫描电镜 ; 细胞凋亡 ; 免疫组织化学 ; Caspase-3
其他题名: Study on Effects of Mid-long Term Simulated Microgravity and Noises on the Auditory Function of Rats
分类号: R852.22
摘要:

背景:

随着航天事业的蓬勃发展,我国航天进入中长期发展阶段。既往研究发现,在航天过程中噪声和失重因素可损伤听觉系统,导致航天员暂时性甚至永久性阈移,减短航天员的职业寿命,影响航天工作的协调进行,间接增加事故发生的可能性,因此有必要对航天性听损伤进行深入研究。本实验利用大鼠作为模拟航天失重和噪声因素的动物模型,应用听觉电生理检测,结合形态学观察及免疫组织化学技术,了解失重、噪声及其复合因素对大鼠听觉系统的影响,观察上述因素在模拟中长期航天任务不同时间点对听觉系统的损伤效应及特点并探讨其损伤机制,为研究航天性听损伤的防治提供理论依据。

目的:

本实验通过使用模拟航天失重和噪声复合因素下的大鼠模型,检测听性脑干反应(auditory brainstem response, ABR)阈值的变化,结合免疫荧光、扫描电镜、免疫组织化学技术等方法,检测听觉电生理的改变,观察大鼠耳蜗毛细胞形态学变化及内耳细胞凋亡因子天冬氨酸特异酶切的半胱氨酸蛋白酶-3(Cysteinyl aspartate specific proteinase-3, Caspase-3)的表达,旨在研究模拟中长期航天失重和噪声及其复合因素对大鼠听觉系统的影响,并探讨其损伤机制。

方法:

将32只雄性SD大鼠随机分为4组:对照组,失重组,噪声组和失重+噪声组,每组各8只。失重组大鼠采用头低足高位尾吊法模拟失重,每日尾吊24小时,持续2周;噪声组大鼠置于声压级为72±2 dB(A)稳态噪声的声场中,每日暴露8小时,连续暴露2周后置于脉冲噪声声场中给予3次峰值声压级为160 dB(A)的脉冲噪声;失重+噪声组大鼠同时暴露于上述两种环境中;对照组大鼠常规饲养2周。实验结束后,检测各组大鼠ABR阈值,取耳蜗标本行免疫荧光及扫描电镜观察毛细胞的形态学改变。

(2)将36只雄性SD大鼠随机分为6组:对照组,3天组,1周组,2周组,4周组和8周组,每组各6只。各实验组大鼠分别于模拟航天失重和噪声的复合因素中暴露相应时间,暴露方法同上。实验结束后,检测各组大鼠ABR阈值,取耳蜗标本行HE染色并检测凋亡因子Caspase-3的表达。对照组大鼠常规饲养,分别于3天、1周、2周、4周和8周时检测其ABR阈值,并于暴露8周后行耳蜗HE染色并检测内耳细胞凋亡因子Caspase-3的表达。

结果:

实验后,失重组、噪声组和失重+噪声组大鼠ABR阈值分别为:23±4dB、57±21dB和79±13dB,均较对照组(14±6dB)明显增高,其中失重+噪声组大鼠ABR阈值明显高于其他各组(P<0.05)。免疫荧光结果显示,失重组大鼠耳蜗损伤以内毛细胞核缺失为主,噪声组大鼠耳蜗损伤以外毛细胞核肿胀及缺失为主,失重+噪声组大鼠耳蜗内、外毛细胞胞核同时存在上述损伤。扫描电镜结果显示,失重组大鼠耳蜗损伤以内毛细胞静纤毛的倒伏及缺失为主,噪声组大鼠耳蜗损伤以外毛细胞静纤毛的倒伏及缺失为主,失重+噪声组大鼠耳蜗内、外毛细胞同时存在上述损伤。对照组免疫荧光及扫描电镜结果未见明显异常。

实验后,3天组、1周组、2周组、4周组和8周组大鼠ABR阈值分别为:79±5dB、83±11dB、85±5dB、90±4dB和80±5dB,均较对照组(13±6dB)明显增高,其中4周组大鼠ABR阈值最高(P<0.05)。耳蜗HE染色结果显示,实验组大鼠耳蜗出现Corti器连续性中断、血性渗出物、螺旋韧带撕裂等损伤。免疫组织化学结果显示,各实验组大鼠内耳细胞中Caspase-3均呈阳性表达,4周组大鼠内耳细胞Caspase-3表达的积分光密度值明显高于其他各组(P<0.05)。对照组大鼠耳蜗HE染色未见明显异常,仅个别内耳细胞Caspase-3呈较弱的阳性表达。

 

结论

(1)模拟航天失重、噪声及两者复合因素均可导致大鼠听功能损伤,其中复合因素所致损伤最重。

(2)模拟航天失重、噪声及两者复合因素均可导致大鼠耳蜗毛细胞结构损伤,其中模拟失重损伤以内毛细胞为主,噪声损伤以外毛细胞为主,复合因素可致内、外毛细胞均出现损伤。

(3)模拟航天失重和噪声复合因素可导致大鼠听功能受损,以4周时最重;

(4)模拟航天失重和噪声复合因素可导致内耳细胞凋亡因子Caspase-3表达上调,以4周时最明显,8周时有所下降。

英文摘要:

Background:

   With the vigorous development of the manned space program, China's space program has came into -long term development stage. Previous studies have found that, the influence of noise and microgravity to the auditory system is larger. And astronauts’ threshold will produce a temporary threshold shift or permanent threshold shift leading to hearing loss to whom exposed to manned spacecraft environment which can shorten astronauts’ career longevity and increase the likelihood of accidents indirectly. So it is necessary to do further research on damage of auditory system in space. In the present study, we used rats as animal models contains simulated microgravity and noises, using auditory brainstem response test and immunohistochemistry to study the influence of microgravity and noises to auditory system of rats. Aimed to observe the damage effect and feature of auditory function, and discuss the damage mechanism in space, to provide theoretical basis for the prophylaxis and treatment of hearing loss in space.

Objectives:

   We establish a rat model of hearing loss in microgravity and noise in manned spacecraft. Observe changes in the inner ear of the model by ABR test, the SEM, Caspase-3 index and apoptosis and necrosis of morphology, in order to explore the specific mechanisms of damage to auditory function of the -long term simulated microgravity and noises environment and discuss the damage mechanism.

Methods

 (1) Thirty-two male SD rats, were randomly divided into four groups: control group, microgravity group, noises group and microgravity + noises group (n=8). The microgravity group was exposed to -30° head down tilt as simulated microgravity with the tail hanging 24 hours a day for 2 weeks. The noises group was exposed in noises including 72±2 dB(A) stationary noise and impulse noise up to 160 dB(A) of 3 times, and the stationary noise was exposed 8 hours a day for 2 weeks, then after 2 weeks’ exposure, exposed to impolse noise 3 times. The microgravity+noises group was exposed to compound environment above. The control group was breeding conventional for 2 weeks. After exposure, ABR tests were performed and the rats were killed, and the cochleas were harvested for immunofluorescence staining and scanning electron microscopy (SEMs) observation.

 (2) Thirty-six rats were randomly divided into 5 experimental groups (3-days, 1-week , 2-weeks, 4-weeks, 8-weeks, n=6) and a control group (n=6). The experimental groups as simulated spaceship environment including microgravity and noises. The intervention methods were the same as above. Immediately after the impulse noise exposure, bilateral ABR thresholds were tested, and the rats were killed and cochleas were harvested for morphological observation and detection of cell apoptosis factors. The control group kept in normal conditions without exposure to microgravity and noise. Bilateral ABR thresholds were tested at corresponding time points, and the rats were killed at 8 weeks, cochleas were harvested for examinations above.

Results

 (1) After the experiment, the ABR threshold in microgravity group, noises group and microgravity + noises group were 23±4 dB, 57±21 dB and 79±13 dB, all increased when compared to the control group and microgravity+noises group has the highest ABR threshold(P<0.05). Immunofluorescence showed hair cell nuclear missing or swelling in the experimental groups, in which the microgravity+noises group appeared worst. SEM showed varying degrees of lodging or missing of cilia of the hair cells. In which, weightlessness mainly led to inner hair cell damage, while combined noise mainly led to outer hair cell damage. The weightlessness + noise group has both damage in inner hair cells and outer hair cells. The control group has no obvious abnormal.

 (2) After exposed to combined environmental conditions, the ABR thresholds of the 3-days group, 1-week group, 2-weeks group, 4-weeks group and 8-weeks group were 83±11 dB, 85±5 dB, 90±4 dB and 80±5 dB respectively. All of the ABR thresholds in the experimental groups were significantly increased compared to the control group (13±6 dB), in which 4 week group showed the highest(P<0.05). Histologically, the experimental groups have fuzziness cochlear structure, compared to the control group. Immunohistochemical staining showed that Caspase-3 expressions in experimental groups were positive in inner ear cells. The tendency of the Caspase-3 expression was in accordance with the ABR threshold in each experimental group. IOD Semiquantitative analysis of the immunohistochemical staining showed that the expression of Caspase-3 in the 4 weeks group was much higher than the other groups(P<0.05). The control group has no obvious abnormal besides individual inner ear cell show weak positive expression of Caspase-3.

Conclusion

 (1) Simulated microgravity, noises and compound factors might cause the damage of  hearing functions of the rats and the compound factors lead to the severest damage .

 (2) Structural damage of hair cells in the inner ear was induced after exposed to simulated microgravity and noises. In which, weightlessness mainly led to inner hair cell damage, while combined noise mainly led to outer hair cell damage.

(3) The compound factors of simulated microgravity and noises might cause the damage of  hearing functions, which was most apparent at 4 weeks.

(4) Expression of caspase-3 in the inner ear was increased after exposed to -long term simulated microgravity and noises, which was most apparent at 4 weeks, and reduced by 8 weeks.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125289
Appears in Collections:北京大学解放军306医院教学医院_学位论文

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作者单位: 北京大学解放军306医院教学医院

Recommended Citation:
陈娜. 模拟中长期航天失重和噪声环境对大鼠听觉系统影响的研究[D]. 北京大学解放军306医院教学医院. 北京大学. 2016.
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