个体特质与生物标志物应激反应模式相关性研究

发布时间：2018-01-01 09:24

本文关键词：个体特质与生物标志物应激反应模式相关性研究　出处：《东南大学》2016年博士论文　论文类型：学位论文

【摘要】：应激是机体对环境刺激具有非特异性的生物学反应现象。应激对健康、行为及工作效率的影响已成为国际上重要的职业卫生问题之一,所以评价应激程度具有非常重要的意义。各种内外环境因素及社会、心理因素刺激均可以使人体或动物产生应激反应,这种反应会在不同程度上引起认知能力的改变。有文献研究表明,情绪稳定性作为一种人格特征,存在明显的个体差异,同时对个体具有相对稳定性。这种稳定性既有认知评价、人格特征、气质类型等个性因素,也有生理学基础。随着近代生命科学的发展,研究工作者对应激认识从早期的现象观察和描述,逐渐发展为从“心理-神经内分泌-行为”多学科多角度,更加系统全面地揭示应激的生理与心理响应特征,及应激对大脑、心理与行为活动影响的深层机制。应激生物标志物检测研究一直是生理学、医学、运动学、心理学研究等所关注的重点。深入研究基于应激标志物的评估方法及应激响应规律,对预防各类应激损伤有重要意义。目前,应激评估方法主要有问卷调查法、行为观察法、电生理、生化及脑成像检测法等多种方法。其中,基于生化检测的评估方法具有客观、准确的优点。但是,对生物样本中的神经活性物质进行定量分析需要非常灵敏、精密的分析方法,这已成为相关研究顺利开展的关键环节。本文选取无创样本唾液,并以唾液α一淀粉酶和皮质醇等为目标物,深入探究其作为应激标志物在认知神经科学等相关研究中的可靠性和有效性。本文主要开展了以下六个主要内容的研究工作：(1)应激研究技术平台的建立及优化；(2)多参数评价应激对小鼠脑与行为的影响；(3)运动对应激标志物的影响初步研究；(4)多参数评价人体心理应激研究；(5)个体特质在心理应激中的调节作用；(6)应激对注意调控能力的影响。具体工作如下：一、应激研究技术平台的建立及优化(1)生物标志物快速检测技术的构建及优化①化学发光免疫法快速测定前白蛋白方法的建立：将聚乙烯亚胺(PEI)修饰的磁颗粒(MBs)通过戊二醛偶联抗体(Nb1),所形成的免疫磁珠(MB/PEI/Nb1)能够特异的结合前白蛋白(PA)形成免疫复合物。该复合物可与包被有辣根过氧化物酶(HRP)和检测抗体(Nb2)的SiO2纳米粒子(HRP-SiO2-Nb2)形成免疫夹心复合物(MB/PEI/Nb1-PA-Nb2/SiO2-HRP)。复合物中的HRP催化鲁米诺、对碘苯酚(PIP)及H2O2反应,提高化学发光(CL)信号。该方法的检测限为0.01μgL-1,线性范围0.05到1000μg L-1,完成整个操作只需40分钟。②六种单胺类神经递质的高效液相色谱-荧光检测法的优化：6种目标物分别为去甲肾上腺素(NE)、多巴胺(DA)、3,4二羧基苯乙酸(DOPAC)、3-甲基-4-羟基苯乙二醇(MHPG)、5-羟色胺、5-羟吲哚乙酸(5-HIAA),采用CosmosilC18 (4.6x250 mm,5μm)色谱柱,流动相：0.1mol/L的醋酸钠溶液：甲醇(体积比5：95),pH为6,其中含0.2mmol/L乙二胺四乙酸二钠；流速2.0 mL/min;在2-300pmoL范围内,目标物具有较好的线性关系(0.999),每种神经递质的回收率为95%-102%。优化后的方法适用于脑组织不同区域的单胺类神经递质检测。③唾液α-淀粉酶(sAA)流动注射分析方法优化：在本组前期研究的基础上进行进一步优化条件,建立了更加快速、简便的sAA流动注射光度测定法。该方法测定一个样本的周期仅为34秒,可以实现自动化操作,精密度高,避免人工操作上时间控制不当所导致的测定差异大的缺点。④其他检测方法的建立：基因检测方法、近红外脑成像检测技术。(2)应激模型的构建及优化应激模型是研究应激与神经内分泌、行为关联性的重要手段。因为大部分研究都是建立动物模型,因此人类数据有限,但啮齿类动物和非人灵长类的一些研究数据,可以应用于人体模型。通过文献总结,我们分别建立了针对动物和人体应激相关的模型,包括小鼠应激模型和人体应激模型。(3)应激评价方法的构建为了考察应激对机体的影响,本课题选择了多种实验范式以测评动物及人体行为。针对小鼠的有：高架十字迷宫、摄食抑制行为、强迫游泳实验、社交行为实验；针对人体的有：Stroop实验、心算评价、眼动实验等。二、多参数评价应激对小鼠脑及行为的影响束缚应激是研究早期事件对行为改变广泛应用的模型之一。但是束缚应激能否引起社交焦虑行为,运动是否能够起到有效干预作用尚存有争议。因此,我们对1月龄小鼠分别进行了每天两小时,共两周的束缚应激试验研究。结果表明,相对于正常控制组,束缚应激小鼠(出生后28天-37天)焦虑和抑郁行为明显提高。此外,束缚应激小鼠行为改变还伴随其海马HDACsmRNA表达和不同脑区神经递质水平的改变。有氧锻炼有效的缓解了束缚应激导致的单胺类神经递质、HDACs mRN A表达和多种行为的改变。研究结果说明束缚应激会导致社交及情绪相关行为的改变,而运动干预会起到缓解作用。三、运动对应激标志物的影响初步研究本文主要探讨儿童在运动应激条件下,sAA活力指标的变化规律,考察其作为一种客观的评价运动应激的生物学参数的可能性。被试为120名小学生。在运动应激条件下,采集他们测试前30分钟、测试结束后即刻及测试结束休息30分钟后的唾液样品。利用流动注射法测定sAA的浓度。本研究的结果显示,被试的sAA水平在运动应激的不同阶段之间无显著性差异,但在不同年级分组之间明显存在个体差异；结合问卷分析,我们推测sAA活力水平可能受到人格对应激的评估和调节作用。流动注射快速测定sAA的方法在客观评价运动应激方面有潜在应用价值。四、多参数评价人体心理应激研究探讨在心理应激压力下,sAA指标与心理应激的相关性,考察其作为一种客观的评价心理应激的生物学参数的可能性。被试为44名研究生(男女生各22名),年龄为23-27岁。在Stroop测试、心算测试心理应激条件下,采集他们测试前10分钟、测试结束时以及测试结束休息10分钟后的唾液样品。利用流动注射法测定sAA的浓度。结果显示被试的sAA水平在心理应激实验时明显变化,也表明流动注射快速测定sAA的方法可用于客观评价心理应激。五、个体特质在心理应激研究中的调节作用为了验证TPQ问卷中的3个人格维度是否与应激的神经内分泌反应相关,我们招募了64名健康本科学生,通过填写TPQ问卷,将全部被试分别分为猎奇(NS)、危害避免(HA)及奖赏效应(RD)值高低不同的两组。所有被试均参加Stroop联合心算的应激测试并测定心率变异性和sAA指标,统计分析分析比较组间差异。结果表明：①高NS组Stroop完成数量显著高于低NS组；高NS、HA组Stroop测试平均反应时显著高于低NS、HA组。②Stroop测试后sAA活力水平显著高于测试前的安静值；心算后高HA组的sAA水平显著高于低HA组。③心率变异性中的Stroo P前均一化低频成分Stroop前(LFnul),均一化化高频成分(HFnul),低频／高频(LF/HF1), srtoop后相邻RR间期差值的均方根(RMSSD2),均一化化低频(HF2)、心算后即刻高频成分(HF3),相邻正常R-R间期差值均方根(RMSSD3)等指标高NS、RD组显著高于低NS、RD组。结果显示不同人格维度与sAA活力和心率变异性参数间存在相关性,对人格的生物基础提供了理论支持。结果说明TPQ人格中的猎奇和奖赏维度可能是影响应激响应特征的重要调节因子。六、应激对注意认知调节能力的影响视觉注意的认知调控常用于运动员技战术的日常训练,但在比赛中采用这种策略却很难奏效。本研究深入探索了高水平重剑运动员和普通大学生在面对模拟对手进攻时视觉注意线索的变化特点。学生组随机分为应激组和控制组,应激组接受冷水刺激。刺激后,检测所有被试对模拟对手进攻的视觉注意眼动规律,并检测sAA及唾液皮质醇指标。结果发现,专业运动员有更高效的首注视点注意效率。应激降低了普通大学生视觉注意的认知调节能力。唾液应激标志物可以作为评价不同特质个体视觉注意控制能力的潜在指标,评价注意认知调节水平。
[Abstract]:Stress is the body of environmental stimulation biological reaction phenomenon is nonspecific. Effects of stress on health, behavior and work efficiency has become one of the important international occupation health problems, so the evaluation of the degree of stress has very important significance. All kinds of internal and external environment factors and social factors, psychological stimulation can make human or animal stress reaction this reaction, will cause the changes of cognitive ability in different extent. A literature research shows that emotional stability as a personality characteristic, the obvious individual differences, also has the relative stability of the individual. The stability of both cognitive evaluation, personality, temperament and personality factors, but also the basis of Physiology. With the development of in modern life science, researchers stress understanding from the phenomenon observed and described earlier, gradually developed from "Psycho neuroendocrine "Multi-disciplinary perspective, more systematically reveal the physiological characteristics and the psychological stress response, and stress on the brain, deep influence mechanism of psychological and behavioral activities. The stress of biomarker detection has been physiology, medicine, kinematics, psychology and other key concern. Further research on evaluation method of stress markers and based on the stress response, has important significance for the prevention of various types of stress damage. At present, stress evaluation methods mainly include questionnaire, behavioral observation, electrophysiological, biochemical methods and brain imaging detection method. The evaluation method of biochemical detection based on an objective, accurate. However, quantitative analysis need to be very sensitive to neural active substances in biological samples, sophisticated analytical methods, it has become a key link in related research carried out smoothly. This paper selects noninvasive saliva samples, And to the alpha amylase and salivary cortisol as a target to explore the reliability as stress markers in related research in cognitive neuroscience and effectiveness. The research work of this paper mainly includes the following six main contents: (1) establish and optimize the stress of technology platform; (2) effect the parameters of stress evaluation on brain and behavior; (3) a preliminary study on the influence of exercise stress; (4) to study the human psychological stress evaluation parameters; (5) individual role in mental stress; (6) pay attention to the impact of stress on the regulatory capacity. The specific work is as follows: 1. Establish and optimize the stress research platform (1) biomarkers for rapid detection of prealbumin determination techniques of construction and optimization of the chemiluminescence immunoassay: polyethyleneimine (PEI) modified magnetic particles (MBs) by Glutaraldehyde coupling antibody (Nb1), formed by immunomagnetic beads (MB/PEI/Nb1) can be combined with specific prealbumin (PA) formation of immune complexes. The complexes can be coated with horseradish peroxidase (HRP) and antibody (Nb2) SiO2 nanoparticles (HRP-SiO2-Nb2) formed a sandwich assay (MB/PEI/Nb1-PA-Nb2/SiO2-HRP). HRP catalyzed Lumino in the compound of iodine, phenol (PIP) and H2O2 reaction, enhance the chemiluminescence (CL) signal. The detection limit is 0.01 gL-1, the linear range of 0.05 to 1000 g L-1, the entire operation was completed in just 40 minutes. The optimization of high performance liquid phase six monoamine neurotransmitters HPLC - fluorescence detection method: 6 targets were norepinephrine (NE), dopamine (DA), 3,4 (DOPAC) two carboxyl phenyl acetic acid, 3- methyl -4- hydroxy phenylglycol (MHPG), 5- serotonin, 5- hydroxyindoleacetic acid (5-HIAA), using CosmosilC18 (4.6x250 mm, 5 M) column, mobile phase: 0.1mol/L sodium acetate solution: methanol (volume ratio 5:95), pH was 6, including two 0.2mmol/L EDTA sodium 2 mL/min; flow rate; in the range of 2-300pmoL, the object has a good linear relationship (0.999), the recovery rate for each neurotransmitter 95%-102%. optimization method after the application of monoamine neurotransmitters in different brain region detection. The salivary amylase (sAA) optimization method for flow injection: to optimize the conditions based on the previous research, the establishment of a more rapid, sAA flow injection spectrophotometric method for the determination of the simple cycle. The method for the determination of a sample only 34 seconds, can realize automatic operation, high precision, avoid the manual operation on time control caused by improper determination of differences. The establishing of defect detection method: gene detection method, near infrared brain As the detection technology. (2) stress model and optimization model of stress and stress is an important means of neuroendocrine, behavior related. Because most of the research is to establish the animal model of the human data is limited, but animal rodents and non-human primates some research data can be applied to the human body through the model. The literature review, we developed for animal and human stress related models, including the model and the stress model of stress in mice. (3) stress evaluation method constructed in order to investigate the effects of stress on the body, this paper selects the various experimental paradigms to assess animal and human behavior. In mice: the elevated plus maze, inhibition of feeding behavior, forced swimming test, social behavior experiment; in view of the human body are: Stroop experiment, mental evaluation, eye movement experiment. Two, multi parameter evaluation of mouse brain and stress The effect of restraint stress is one of the early events of the widely used model of behavior change. But the restraint stress can cause anxiety and social behavior, whether exercise can play a role in effective intervention remains controversial. Therefore, we are on January old mice two hours a day, experimental study on restraint stress for two weeks. The results showed that and compared with the normal control group, restraint stress mice (postnatal 28 days -37 days) anxiety and depression behavior improved significantly. In addition, restraint stress mice behavior change with the change of the expression of HDACsmRNA in hippocampus and brain neurotransmitter levels. Aerobic exercise can effectively relieve the monoamine neurotransmitters in restraint stress caused by HDACs mRN the expression of A and a variety of behavioral changes. Research results indicate that restraint stress can lead to social and emotional behavior change, and exercise intervention will play a role in mitigation. Three, the preliminary study on the influence of this paper mainly discusses the children in the exercise stress conditions exercise stress signs, changes of sAA activity index, investigate the possibility as biological parameters of an objective evaluation of exercise stress. The subjects were 120 students. In the exercise stress conditions, they collected 30 minutes before the test. After the end of the test and at the end of the test after 30 minutes of rest of the saliva samples. SAA concentrations were determined by flow injection method. The results of this study showed that there was no significant difference between subjects sAA levels at different stages of exercise stress, but there are obvious individual differences among different grade groups; combined with the analysis of the questionnaires, we speculate that the level of sAA activity may be evaluated on stress and personality regulation. Rapid determination method of sAA flow injection has the potential application value in the evaluation of exercise stress. More than four. The study of human psychological stress parameters to evaluate the in psychological stress, correlation between sAA index and psychological stress, to investigate the possibility as biological parameters of an objective evaluation of the psychological stress. The subjects were 44 students (male and female 22), aged 23-27 years old. In the Stroop test, the psychological stress condition under the mental test, they collected 10 minutes before the test, the end of the test and the test ended after 10 minutes of rest of the saliva samples. SAA concentrations were determined by flow injection method. The results showed that the level of sAA was changed in psychological stress experiment, also shows that the method of quick determination of sAA flow injection can be used to evaluate the psychological stress. Five, regulation of individual characteristics in the study of psychological stress in order to verify whether the 3 personality dimensions of TPQ questionnaire related to the neuroendocrine response to stress, we recruited 64 healthy the The students by filling out TPQ questionnaire, all subjects were divided into Adventures (NS) (HA), to avoid harm and reward effect (RD) values of different two groups. All subjects are in Stroop United stress test and determination of heart rate variability and sAA index, statistical analysis differences between groups. The results showed: high NS groups of Stroop number was significantly higher than that of low NS group; high NS, HA group Stroop test average reaction time was significantly higher than that of low NS, HA group. SAA activity level of Stroop was significantly higher than that before the test after the test value after sAA quiet; mental water high HA group was significantly higher than that of low HA group. The heart rate variability in Stroo P before homogenization of low-frequency components before Stroop (LFnul), the homogenization of high frequency components (HFnul), low frequency / high frequency (LF/HF1), srtoop after the adjacent RR interval difference of root mean square (RMSSD2), the homogenization of the low frequency (HF2), immediately after the high frequency components (mental arithmetic HF3), adjacent Normal R-R interval difference of root mean square (RMSSD3) and other indicators of RD group was significantly higher than that of high NS, low NS, RD group. The results showed that the correlation between different personality dimensions and sAA activity and heart rate variability among the parameters, provides a theoretical support for the biological basis of personality. The results show that the TPQ personality curiosity and reward dimension may be the important regulatory factors affecting stress response characteristics. Six, stress on the attention of visual attention cognition regulation used in the daily training of athletes'tactics influence cognitive regulation ability, but in the game using this strategy is hard work. This study explores the deep into the high level epee athletes and college students in the face of simulated opponents at visual attention characteristics of clues. The students were randomly divided into stress group and control group, stress group received cold stimulation. After stimulation, detection of all subjects attack on the opponent's attention to eye visual simulation The dynamic law, and detection of sAA and salivary cortisol index. The results showed that professional athletes are the first focus the attention of more efficient efficiency. Stress reduces the visual attention of ordinary college students cognitive regulation ability. Saliva stress markers can be used as a potential indicator control capability evaluation of different individual characteristics of visual attention, pay attention to adjust the level of cognitive evaluation.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R33

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