大鼠边缘下区NR2B，BDNF和Caspase-3在条件性恐惧消退保持过程中的作用研究

发布时间：2018-05-11 03:35

本文选题：边缘下区 + NR2B　；参考：《第三军医大学》2010年硕士论文

【摘要】： 背景:近年来,随着各种突发灾难性事件(地震、洪水、恐怖袭击、车祸等)的发生,创伤后应激障碍(post-traumatic stress disorder,PTSD)成了关注的重点。所谓PTSD是亲身经历或者目睹异乎寻常的威胁性、灾难性事件后发生的一系列生理、心理应激所表现出来的临床综合症。创伤记忆一旦形成,就很难消退,给患者带来很大痛苦。目前临床上治疗PTSD常用的非药物方法为暴露疗法(基于消退原理的行为治疗方法),但约有40%的病人疗效难以维持,表现为已消退的恐惧记忆很快再现。这提示消退保持障碍可能是PTSD发生和治疗困难的关键。因此,研究恐惧消退保持的神经生物学机制具有重要意义。目前,相对于条件性恐惧的获得、巩固与表达过程,对恐惧消退机制的研究尚很不够,许多问题有待阐明。恐惧消退是指当条件性刺激(conditioned stimulus,CS)重复呈现而缺乏非条件刺激(unconditioned stimulus,US,如足底电击)配对呈现,所致的条件性恐惧反应的逐渐减弱或消失。它不是抹掉了先前形成的CS-US联结记忆,而是形成了CS-no US的新记忆(消退记忆)。大量研究表明内侧前额叶(medial prefrontal cortex,mPFC)边缘下区(infralimbic cortex,IL)参与了条件化恐惧的消退过程,在消退保持中起重要作用,是消退记忆长期保持的场所。但是IL区在消退记忆长时保持中的作用机制如何?哪些物质参与了消退过程尚不清楚。我们在以往研究的基础上,选用条件性恐惧模型和恐惧消退模型,综合行为学、形态学和Western-Blot方法对大鼠IL区在恐惧消退保持中的作用及机制进行探索,以期找到条件性恐惧消退的最佳“时间窗”和关键分子,探讨IL区在声音条件性恐惧消退保持中的作用机制,为提高PTSD治疗效果提供依据。目的:探讨给予条件性恐惧消退训练后,大鼠边缘下区NR2B、BDNF以及caspase-3在消退保持中的动态变化。方法:75只SD大鼠随机分成空白对照组(Cont组,无任何处理),消退对照组(N-EXE组,无消退训练)和24h消退组(EXE组)。采用声音-足底电击配对刺激建立条件性恐惧模型,并于恐惧建立24h后进行消退训练,比较消退训练后第1、3、7天大鼠的僵立行为变化以及IL区NR2B、BDNF以及caspase-3的变化。结果: 1.各组大鼠消退保持成绩的变化: 消退训练后,消退组大鼠消退保持成绩逐渐增加(恐惧逐渐减少)。消退训练后第3天和7天,24h消退组消退保持成绩好于消退对照组。(p0.01)。 2.各组大鼠IL区NR2B、BDNF免疫反应阳性物质的变化: (1)消退对照组和24h消退组大鼠IL区NR2B和BDNF含量均逐渐增加,第1、3.天增加迅速,第7天最高,但均低于空白对照组,差异显著(p0.01); (2)消退对照组和24h消退组相比较:第1天两组无明显差异;第3、7天两组差异显著,且24h消退组显著高于消退对照组(p0.05)。 3.各组大鼠mPFC NR2B以及BDNF蛋白含量的变化: 消退对照组和24h消退组大鼠在消退后第1、3、7天IL区NR2B、BDNF蛋白含量逐渐增加,第1、3天增加迅速,消退对照组第7天基本恢复正常。在各时间点,消退对照组NR2B和BDNF的蛋白含量显著低于24h消退组和空白对照组。 4.各组大鼠IL区caspase-3免疫反应阳性物质的变化: 与空白对照组比较,(1)消退对照组和24h消退组大鼠IL区caspase-3免疫反应阳性物质量明显高于空白对照组;消退训练后第7天,24h消退组caspase-3免疫反应阳性物质量接近空白对照组水平;(2)消退训练后随着时间的增加,消退对照组和24h消退组caspase-3含量逐渐减少,第7天24h消退组caspase-3量基本恢复正常;(3)在各时间点,24h消退组caspase-3量显著低于消退对照组(p0.05)。结论: 1.行为学的结果显示:恐惧消退训练后一周内,消退对照组和24h消退组大鼠的消退保持成绩均逐渐提高(条件性恐惧逐渐改善),24h消退组好于消退对照组。提示:消退训练有利于恐惧记忆的消退。因此,对PTSD患者要给予及时积极的暴露治疗; 2.免疫组化和Western Blot结果显示: IL区NR2B和BDNF的免疫阳性细胞数以及蛋白水平随着消退成绩的改善而逐渐增加,在消退训练后3-7天达最高。提示:消退训练后3-7天可能是恐惧消退保持的关键时段,IL区NR2B和BDNF在恐惧消退保持中起重要作用。 3.消退训练后,随着IL区caspase-3含量逐渐下降,且24h消退组成绩好于消退对照组。提示:caspase-3可能参与了恐惧消退记忆保持过程中IL区的可塑性变化,但其中的机制还需进一步研究。
[Abstract]:Background: in recent years, post-traumatic stress disorder (PTSD) has become a focus with the occurrence of a variety of catastrophic events (earthquakes, floods, terrorist attacks, and car accidents). The so-called PTSD is a series of physiological, psychological stresses that occur after the experience of personal experience or the unusual coerced, disastrous events. The manifestation of clinical syndrome. Once the traumatic memory is formed, it is difficult to recede and bring great pain to the patient. At present, the clinical treatment of PTSD is a non drug method for exposure therapy (based on the behavior therapy based on the principle of regression), but about 40% of the patients are difficult to maintain the curative effect, showing the rapid reappearance of the receded fear memory. The key to the occurrence and treatment difficulties of PTSD may be the key to the difficulty of occurrence and treatment. Therefore, it is of great significance to study the neurobiological mechanism of the decline and retention of fear. At present, there is not enough study on the process of consolidation and expression relative to the conditioned fear, and many problems remain to be clarified.
Fear receding is that the conditional stimulus (conditioned stimulus, CS) is presented repeatedly without unconditional stimulation (unconditioned stimulus, US, such as the foot shock), and the conditioned fear response gradually diminishes or disappears. It does not erase the previously formed CS-US connection memory, but forms a new memory of CS-no US. A large number of studies have shown that the medial prefrontal cortex (mPFC) in the submarginal zone (infralimbic cortex, IL) participates in the fading process of conditioned fear, which plays an important role in the retention and retention, and is the place for the long-term retention of receding memory. But what is the mechanism of the long-term retention of the memory in the IL region? Which material is the material reference? It is not clear that the retreat process is not yet clear.
On the basis of previous studies, we choose the conditioned fear model and the fear elimination model, the comprehensive behavior, the morphology and the Western-Blot method to explore the role and mechanism of the IL region in the retreat and retention of the rat, in order to find the best "time window" and the key elements of the conditioned fear regression, and to explore the sound conditions in the IL region. The mechanism of sexual fear extinction plays a role in improving the therapeutic effect of PTSD.
Objective: To explore the dynamic changes of NR2B, BDNF and Caspase-3 in the submarginal area of rats after conditioned fear extinction training.
Methods: 75 SD rats were randomly divided into blank control group (group Cont, no treatment), the control group (group N-EXE, non regression training) and 24h subsiding group (EXE group). The conditioned fear model was established by the sound and foot shock paired stimulation. After the fear established 24h, the regression training was carried out, and the stiffness behavior of the rats after the regression training was compared. Changes and changes of NR2B, BDNF and Caspase-3 in IL area.
Result:
1. the changes of the regression and retention of the rats in each group:
After the retreat training, the decline and retention of the rats in the subsiding group increased gradually (fear gradually decreased). After the third and 7 days of the retreat training, the decline of the 24h subsided group was better than that of the control group (P0.01).
2. the changes of NR2B and BDNF immunoreactive substances in IL region of each group.
(1) the content of NR2B and BDNF in the IL area of the control group and the 24h subsided group increased gradually, and increased rapidly on the day 1,3., the highest in the seventh day, but all lower than that in the blank control group (P0.01).
(2) the regression control group and the 24h regression group were compared: there was no significant difference between the two groups in first days; on the 3,7 day, the difference between the two groups was significant, and the 24h regression group was significantly higher than that of the control group (P0.05).
3. the changes of mPFC NR2B and BDNF protein contents in rats of each group:
In the regression control group and the 24h regression group, the IL area NR2B, BDNF protein content increased gradually at the day 1,3,7 day after the regression, and the 1,3 day increased rapidly. The decrease of the control group was basically restored to normal at seventh days. At every time point, the protein content of NR2B and BDNF in the control group was significantly lower than that in the 24h regression group and the blank control group.
4. the changes of Caspase-3 immunoreactive substance in IL region of rats in each group.
Compared with the blank control group, (1) the quality of Caspase-3 immunoreactive substance in the IL region of the control group and the 24h regression group was significantly higher than that in the blank control group. The quality of the caspase-3 immunoreactive substance in the 24h subsiding group was close to the blank control group at the seventh day after the retreat training; (2) the regression control group and 24h subsided with the time of elimination training. The content of Caspase-3 decreased gradually, and the amount of Caspase-3 in the seventh day 24h subsided group basically recovered to normal, and (3) at each time point, the caspase-3 amount in the 24h subsiding group was significantly lower than that of the control group (P0.05).
Conclusion:
The results of 1. behavioral study showed that in the week after the fear elimination training, the regressed control group and the 24h regression group were gradually improved (the conditioned fear improved gradually), and the 24h subsided group was better than the regression control group. It suggested that the retreat training was beneficial to the decline of fear memory. Therefore, the patients of PTSD should be given a timely and active exposure. Treatment;
2. immunohistochemical and Western Blot results showed that the number of immunoreactive cells and protein levels of NR2B and BDNF in IL area were gradually increased with the improvement of regression results, and the highest in 3-7 Tianda after the retreat training. It was suggested that the 3-7 day after the retreat training may be the key time for the fear to decline and the IL region NR2B and BDNF play an important role in the decline and retention of fear. Effect.
After 3. regression training, the content of Caspase-3 in IL area decreased gradually, and the result of 24h subsiding group was better than that of the control group. It was suggested that caspase-3 may be involved in the plasticity of IL region during the memory retention of fear, but the mechanism needs further study.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R33

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