本文關(guān)鍵詞：PTSD樣大鼠杏仁中央核核受體MR、GR表達變化的研究　出處：《中國醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 創(chuàng)傷后應(yīng)激障礙(PTSD) SPS 杏仁中央核(CeA) 鹽皮質(zhì)激素受體(MR) 糖皮質(zhì)激素受體(GR)

【摘要】： PTSD樣大鼠杏仁中央核核受體MR、GR表達變化的研究 目的 PTSD--創(chuàng)傷后應(yīng)激障礙(post-traumatic stress disorder，PTSD)是指由于災(zāi)害、戰(zhàn)爭、恐怖事件、交通意外、虐待等引起的巨大痛苦或受驚嚇、遭受悲劇導(dǎo)致的精神、身心癥狀的持續(xù)狀態(tài)或焦慮綜合征。隨著戰(zhàn)爭、社會暴力、重大交通事故和自然災(zāi)害等意外的增多，PTSD發(fā)生率也越來越高�，F(xiàn)今PTSD以其發(fā)病率高、病程長、難以治愈等特點，嚴重影響人們身心健康而備受關(guān)注。 臨床研究發(fā)現(xiàn)，PTSD患者血中糖皮質(zhì)激素濃度(glucocorticoid，GC)異常低下。皮質(zhì)酮主要通過作用于MR(mineralocorticoid receptor，鹽皮質(zhì)激素受體)和GR(glucocorticoid receptor，糖皮質(zhì)激素受體)這兩種核受體發(fā)揮基因轉(zhuǎn)錄調(diào)節(jié)作用。杏仁核是GC的靶點，杏仁中央核(central amygdaloid nucleus，CeA)和皮質(zhì)內(nèi)側(cè)核群(medial amygdaloid nucleus，MeA)都有MR和GR的表達，MR的表達明顯弱于GR。MR和GR的存在提示CeA和MeA與能夠?qū)C的基礎(chǔ)和應(yīng)激水平進行調(diào)節(jié)。這種調(diào)節(jié)作用與海馬及前額皮質(zhì)這些反饋抑制區(qū)的作用有實質(zhì)上的區(qū)別。目前認為杏仁核上的GR對應(yīng)激調(diào)節(jié)發(fā)揮正反饋的作用。為此，我們研究PTSD樣大鼠CeA的MR和GR的變化，為進一步揭示PTSD的發(fā)病機制提供實驗依據(jù)。 實驗方法 1、實驗動物及分組 成年健康雄性Wistar大鼠80只，體重150--180g(中國醫(yī)科大學(xué)實驗動物中心提供)，實驗室條件飼養(yǎng)，隨機分為四組，即無連續(xù)單一應(yīng)激(single prolonged stress，SPS)刺激的對照組及SPS刺激后靜養(yǎng)24h、7d和14d組，其中SPS刺激后7d組為PTSD樣大鼠組。 2、PTSD樣大鼠模型的建立 采用2005年日本文部省召開的國際PTSD科學(xué)會議確定的關(guān)于PTSD樣大鼠模型--SPS刺激后無干擾喂養(yǎng)7天的方法。SPS刺激是指將大鼠連續(xù)進行下述步驟處理：禁錮2h；強迫性游水20min(水深40cm，水溫25℃)；休息15分鐘后乙謎麻醉至意識喪失。 3、免疫組化SABC法檢測CeA的MR和GR的表達 分別取正常及經(jīng)過SPS刺激后24h、7d、14d各組大鼠依次麻醉后灌流固定取腦，續(xù)固定后浸于Holt′s液至沉下。制作14μm厚冰凍切片，進行MR、GR的免疫組化SABC法，然后光學(xué)顯微鏡(OLYMPUS，BX60，Japan)下觀察、攝片，并做圖像分析和統(tǒng)計學(xué)分析。 4、Western Blotting檢測CeA的MR和GR的表達 分別取對照組及SPS刺激后24h、7d、14d各組大鼠的新鮮CeA組織，進行Western Blotting檢測CeA的MR和GR的表達，并做圖像分析和統(tǒng)計學(xué)分析。 結(jié)果 1、免疫組織化學(xué)反應(yīng)結(jié)果 (1)MR的免疫組化結(jié)果： 高倍鏡下可見MR分布于CeA。對照組MR的陽性表達最強，大多分布在神經(jīng)元胞體，但也可見少數(shù)分布于突起；SPS后24h組與對照組相比，MR的陽性表達明顯降低，陽性神經(jīng)元數(shù)量少；SPS后7d組與SPS后24h組相比，MR陽性表達明顯升高，陽性神經(jīng)元數(shù)量增多，并且可見陽性神經(jīng)元的胞體上發(fā)出一些較長的突起，也呈MR陽性表現(xiàn)：SPS后14d組與SPS后7d組相比，陽性神經(jīng)元數(shù)量增多，但陽性表達平均強度略有下調(diào)，并且沒有看到呈MR陽性的神經(jīng)元突起。 (2)GR的免疫組化結(jié)果： 高倍鏡下可見GR分布于CeA：對照組GR的陽性表達最強，分布在神經(jīng)元的細胞核，可見較多強陽性的細胞核；SPS后24h組與對照組相比，GR的陽性表達明顯降低，陽性神經(jīng)元細胞核數(shù)量少；SPS后7d組與SPS后24h組相比，GR陽性表達明顯升高，陽性神經(jīng)元細胞核數(shù)量增多；SPS后14d組與SPS后7d組相比，陽性表達強度未見明顯升高，但強陽性神經(jīng)元細胞核數(shù)量增多。 2、Western Blotting結(jié)果 (1)MR的Western Blotting結(jié)果發(fā)現(xiàn)SPS刺激后大鼠CeA的MR的表達為24h降低、7d、14d時迅速回升。 (2)GR的Western Blotting結(jié)果發(fā)現(xiàn)SPS刺激后大鼠CeA的GR表達為24h降低、7d、14d時漸續(xù)回升。 3、圖像分析及統(tǒng)計學(xué)分析： 經(jīng)SPS刺激后，CeA的MR表達于24h時明顯下調(diào)，而7d時快速回升，至14d時趨于穩(wěn)定，，但仍低于正常狀態(tài)(p＜0.01)；經(jīng)SPS刺激后，CeA的GR表達于24h時急劇下調(diào)，而7d、14d時呈現(xiàn)逐漸回升趨勢，但仍低于正常狀態(tài)(p＜0.01)；經(jīng)SPS刺激后，CeA的MR／GR值于24h時明顯上調(diào)，而7d、14d時呈現(xiàn)下降趨勢，逐漸接近正常狀態(tài)。 結(jié)論 1、PTSD樣大鼠杏仁中央核核受體MR、GR表達變化及MR／GR比值失衡，可能是致HPA軸調(diào)節(jié)紊亂，從而引發(fā)PTSD相關(guān)癥狀的重要因素。 2、PTSD樣大鼠杏仁中央核的神經(jīng)元突起增長、增多，提供了杏仁中央核與PTSD，恐怖增強相關(guān)的形態(tài)學(xué)基礎(chǔ)。
[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.

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R322

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