PTSD样大鼠杏仁中央核核受体MR、GR表达变化的研究

发布时间：2018-01-10 22:24

  本文关键词：PTSD样大鼠杏仁中央核核受体MR、GR表达变化的研究　出处：《中国医科大学》2007年硕士论文　论文类型：学位论文

  更多相关文章： 创伤后应激障碍(PTSD) SPS 杏仁中央核(CeA) 盐皮质激素受体(MR) 糖皮质激素受体(GR)

【摘要】： PTSD样大鼠杏仁中央核核受体MR、GR表达变化的研究 目的 PTSD--创伤后应激障碍(post-traumatic stress disorder，PTSD)是指由于灾害、战争、恐怖事件、交通意外、虐待等引起的巨大痛苦或受惊吓、遭受悲剧导致的精神、身心症状的持续状态或焦虑综合征。随着战争、社会暴力、重大交通事故和自然灾害等意外的增多，PTSD发生率也越来越高。现今PTSD以其发病率高、病程长、难以治愈等特点，严重影响人们身心健康而备受关注。 临床研究发现，PTSD患者血中糖皮质激素浓度(glucocorticoid，GC)异常低下。皮质酮主要通过作用于MR(mineralocorticoid receptor，盐皮质激素受体)和GR(glucocorticoid receptor，糖皮质激素受体)这两种核受体发挥基因转录调节作用。杏仁核是GC的靶点，杏仁中央核(central amygdaloid nucleus，CeA)和皮质内侧核群(medial amygdaloid nucleus，MeA)都有MR和GR的表达，MR的表达明显弱于GR。MR和GR的存在提示CeA和MeA与能够对GC的基础和应激水平进行调节。这种调节作用与海马及前额皮质这些反馈抑制区的作用有实质上的区别。目前认为杏仁核上的GR对应激调节发挥正反馈的作用。为此，我们研究PTSD样大鼠CeA的MR和GR的变化，为进一步揭示PTSD的发病机制提供实验依据。 实验方法 1、实验动物及分组 成年健康雄性Wistar大鼠80只，体重150--180g(中国医科大学实验动物中心提供)，实验室条件饲养，随机分为四组，即无连续单一应激(single prolonged stress，SPS)刺激的对照组及SPS刺激后静养24h、7d和14d组，其中SPS刺激后7d组为PTSD样大鼠组。 2、PTSD样大鼠模型的建立 采用2005年日本文部省召开的国际PTSD科学会议确定的关于PTSD样大鼠模型--SPS刺激后无干扰喂养7天的方法。SPS刺激是指将大鼠连续进行下述步骤处理：禁锢2h；强迫性游水20min(水深40cm，水温25℃)；休息15分钟后乙谜麻醉至意识丧失。 3、免疫组化SABC法检测CeA的MR和GR的表达 分别取正常及经过SPS刺激后24h、7d、14d各组大鼠依次麻醉后灌流固定取脑，续固定后浸于Holt′s液至沉下。制作14μm厚冰冻切片，进行MR、GR的免疫组化SABC法，然后光学显微镜(OLYMPUS，BX60，Japan)下观察、摄片，并做图像分析和统计学分析。 4、Western Blotting检测CeA的MR和GR的表达 分别取对照组及SPS刺激后24h、7d、14d各组大鼠的新鲜CeA组织，进行Western Blotting检测CeA的MR和GR的表达，并做图像分析和统计学分析。 结果 1、免疫组织化学反应结果 (1)MR的免疫组化结果： 高倍镜下可见MR分布于CeA。对照组MR的阳性表达最强，大多分布在神经元胞体，但也可见少数分布于突起；SPS后24h组与对照组相比，MR的阳性表达明显降低，阳性神经元数量少；SPS后7d组与SPS后24h组相比，MR阳性表达明显升高，阳性神经元数量增多，并且可见阳性神经元的胞体上发出一些较长的突起，也呈MR阳性表现：SPS后14d组与SPS后7d组相比，阳性神经元数量增多，但阳性表达平均强度略有下调，并且没有看到呈MR阳性的神经元突起。 (2)GR的免疫组化结果： 高倍镜下可见GR分布于CeA：对照组GR的阳性表达最强，分布在神经元的细胞核，可见较多强阳性的细胞核；SPS后24h组与对照组相比，GR的阳性表达明显降低，阳性神经元细胞核数量少；SPS后7d组与SPS后24h组相比，GR阳性表达明显升高，阳性神经元细胞核数量增多；SPS后14d组与SPS后7d组相比，阳性表达强度未见明显升高，但强阳性神经元细胞核数量增多。 2、Western Blotting结果 (1)MR的Western Blotting结果发现SPS刺激后大鼠CeA的MR的表达为24h降低、7d、14d时迅速回升。 (2)GR的Western Blotting结果发现SPS刺激后大鼠CeA的GR表达为24h降低、7d、14d时渐续回升。 3、图像分析及统计学分析： 经SPS刺激后，CeA的MR表达于24h时明显下调，而7d时快速回升，至14d时趋于稳定，，但仍低于正常状态(p＜0.01)；经SPS刺激后，CeA的GR表达于24h时急剧下调，而7d、14d时呈现逐渐回升趋势，但仍低于正常状态(p＜0.01)；经SPS刺激后，CeA的MR／GR值于24h时明显上调，而7d、14d时呈现下降趋势，逐渐接近正常状态。 结论 1、PTSD样大鼠杏仁中央核核受体MR、GR表达变化及MR／GR比值失衡，可能是致HPA轴调节紊乱，从而引发PTSD相关症状的重要因素。 2、PTSD样大鼠杏仁中央核的神经元突起增长、增多，提供了杏仁中央核与PTSD，恐怖增强相关的形态学基础。
[Abstract]:Study on the changes of the expression of MR and GR in the central nucleus of amygdala of PTSD like rats
objective
Posttraumatic stress disorder PTSD-- (post-traumatic stress disorder, PTSD) is due to disaster, war, terrorist incidents, traffic accident, abuse caused great pain or frightened, the tragedy caused by mental, physical and mental symptoms of persistent or anxiety syndrome. With the war and social violence, more serious traffic accidents and natural disaster accident, the incidence of PTSD is increasing. Nowadays, PTSD with its high incidence rate, long course of disease, the characteristics of difficult to cure, seriously affecting people's physical and mental health.
The clinical study found that patients with PTSD blood glucocorticoid concentration (glucocorticoid, GC). The abnormal low corticosterone mainly by acting on MR (mineralocorticoid receptor, mineralocorticoid receptor (glucocorticoid) and GR receptor, glucocorticoid receptor) of the two nuclear receptor gene play a role in transcriptional regulation. The amygdala is the target of GC, almond the central nucleus (central amygdaloid nucleus, CeA) and cortex medial nucleus (medial amygdaloid nucleus, MeA) have the expression of MR and GR, CeA and MeA indicated that the expression of MR was significantly weaker than that of GR.MR and GR can adjust and on the basis of GC and stress level. The regulation and the hippocampus and prefrontal cortex the role of feedback inhibition is fundamentally different. Now that the amygdala on GR on stress regulation play the role of positive feedback. Therefore, we study the changes of PTSD rats CeA MR and GR, as in One step to reveal the pathogenesis of PTSD provides experimental basis.
Experimental method
1, experimental animals and groups
Healthy adult male Wistar rats 80, weight 150--180g (experimental animal center of China Medical University), laboratory rearing conditions, were randomly divided into four groups, namely single prolonged stress (single prolonged, stress, SPS) and control group SPS stimulation after resting 24h, 7d and 14d group, the 7d group after SPS stimulation PTSD like rats.
2, the establishment of PTSD like rat model
By using the.SPS method on the model of --SPS stimulated PTSD rats after 7 days feeding without interference in 2005 the Japanese Ministry of education at the International Science Conference on PTSD 'stimulus refers to the following steps: continuous rat forced swimming imprison 2H; 20min (40cm water depth, water temperature of 25 DEG C); after 15 minutes of rest B mystery anesthesia to loss of consciousness.
3, immunohistochemical SABC method was used to detect the expression of MR and GR in CeA
Were normal and after SPS 24h, 7d, 14d rats were anesthetized and perfused fixed brains were removed and fixed after immersion in Holt continued to sink. 's liquid produced 14 m thick frozen sections of MR, GR by immunohistochemistry SABC method, and optical microscope (OLYMPUS, BX60, Japan) under observation, radiography, and image analysis and statistical analysis.
4, Western Blotting detection of MR and GR expression of CeA
The fresh CeA tissues of rats in control group and SPS after stimulation, 24h, 7d and 14d were taken respectively, Western Blotting was used to detect MR and GR expression in CeA, and image analysis and statistical analysis were performed.
Result
1, the results of immunohistochemistry
(1) immunohistochemical results of MR:
High magnification MR distribution in the CeA. control group the expression of MR was the strongest, mostly distributed in neurons, but also visible ethnic distribution in neurites; after SPS 24h group compared with the control group, the positive expression of MR was significantly reduced, the number of positive neurons; SPS 7d group and SPS 24h group compared. The positive expression of MR was significantly increased, the number of positive neurons increased, and visible positive neurons on the soma of a longer neurites, showed MR positive expression after 14d: SPS group and SPS 7d group compared to the number of positive neurons increased, but the expression of the average intensity slightly down, and did not see a MR projection neurons.
(2) immunohistochemical results of GR:
High magnification GR distribution in the CeA control group: GR positive expression was the strongest, distributed in the neuronal nucleus, showed more strong positive nuclei; SPS 24h group compared with the control group, the positive expression of GR positive neurons decreased obviously, the number of nuclei; SPS 7d group and SPS 24h group compared. The positive expression of GR was significantly increased, the number of positive neurons in nucleus increased after SPS; 14d group and SPS 7d group compared with the positive expression intensity had no obvious increase, but the number of positive nuclei of neurons increased.
2, Western Blotting results
(1) the Western Blotting results of MR found that the MR expression of CeA in rats was decreased after SPS stimulation, and the 7d and 14d increased rapidly.
(2) the Western Blotting results of GR found that the GR expression of CeA in rats was decreased after SPS stimulation, and the 7d and 14d increased gradually.
3, image analysis and statistical analysis:
After SPS stimulation, CeA MR expression was down regulated in 24h, and 7d fast recovery to 14d tends to be stable, but still lower than normal (P < 0.01); after SPS stimulation, CeA GR expression in 24h sharply, and 7d, 14d showed a gradual upward trend, but is still lower than the normal (P < 0.01); after SPS stimulation, MR / GR value of CeA in 24h was significantly increased, while 7d and 14d showed a downward trend, gradually close to the normal state.
conclusion
1, the expression of MR and GR and the imbalance of MR / GR ratio in the central nucleus of amygdala in PTSD like rats may be the important factors that cause the disorder of HPA axis and lead to PTSD related symptoms.
2, the neurites of the central nucleus of the amygdala in PTSD like rats increased and increased, providing a morphological basis for the enhancement of the central nucleus of the almond and PTSD, and the enhancement of terror.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R322

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