發(fā)布時間：2018-06-12 01:13

  本文選題：下丘腦 + 束縛　； 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:應激是機體通過神經(jīng)、內(nèi)分泌以及其他系統(tǒng)共同參與調(diào)節(jié)的一種全身非特異適應性反應。適度的應激可以增加機體抵抗外界危險因素的能力,但過度應激也可造成機體的損傷,影響機體正常的心理、生理及代謝功能。精神、心理應激事件在生活中隨處可見,在法醫(yī)學檢案中時常遇到被羈押的犯罪嫌疑人或遭受重大生活事件的當事人發(fā)生快速意外死亡的案例,經(jīng)尸表檢驗無明顯損傷,經(jīng)系統(tǒng)解剖無特異性病理改變,因此死因難以確定。認真分析上述案件的背景不難發(fā)現(xiàn)存在一個共同的特點,即均存在明確的心理應激狀況。通過系統(tǒng)解剖在排除所有死因的前提下,目前部分學者認為這類死亡為應激性死亡,但尚缺乏足夠的科學依據(jù),還有待于進行實驗研究給予支持。下丘腦是應激反應的重要調(diào)節(jié)中樞,因此本研究在成功建立不同時程束縛加冰水游泳應激大鼠模型的基礎(chǔ)上,系統(tǒng)觀察了下丘腦組織病理學改變、內(nèi)質(zhì)網(wǎng)應激信號通路相關(guān)蛋白表達變化及糖皮質(zhì)激素水平變化,旨在探討應激導致的下丘腦神經(jīng)細胞損傷是否有內(nèi)質(zhì)網(wǎng)應激的參與,為應激狀態(tài)下死亡機制的研究提供病理形態(tài)學依據(jù)。方法:1本實驗將100只體重為(200±20)g SD雄性大鼠隨機分組,分為1天、3天、7天、14天和21天組,每組大鼠10只,并設(shè)定相應時間點的各正常對照組10只。采用將大鼠仰臥位固定每日6小時(8:00-14:00)的束縛處理,同時禁食水,束縛結(jié)束后進行5分鐘冰水游泳。應激各組進行束縛處理時,正常對照組大鼠于飼養(yǎng)籠中,同樣禁食水6小時,其余時間各組大鼠自由進食、飲水。各實驗組及對應對照組大鼠于實驗結(jié)束第二天處死,取血留血清做ELISA檢測,腦組織經(jīng)固定、脫水、透明、包埋、連續(xù)切片等處理后待做各種染色使用。1)記錄大鼠體重變化。2)硫堇特殊染色觀察神經(jīng)細胞尼氏體變化。3)Fluoro-jade B染色觀察神經(jīng)細胞變性壞死情況。4)酶聯(lián)免疫吸附試驗檢測血清中糖皮質(zhì)激素濃度變化。5)he染色觀察腎上腺病理學改變。6)免疫組織化學染色觀察ers相關(guān)蛋白表達變化。2陽性細胞比例及統(tǒng)計學分析:運用全景組織細胞定量分析系統(tǒng)對免疫組化實驗中陽性細胞(棕色神經(jīng)細胞)比例進行統(tǒng)計,目前此軟件用于組織細胞計數(shù)已在國外得到廣泛認可。采用spss21.0統(tǒng)計軟件進行統(tǒng)計學分析,各組均數(shù)的比較行單因素方差分析(anova),用最小顯著差法(leastsignificantdifference,lsd)作兩兩比較,以p0.05為有顯著統(tǒng)計學差異。結(jié)果:1大鼠體重變化結(jié)果隨時間的延長,對照組大鼠體重呈上升趨勢。與對照組相比,實驗1天組大鼠體重略有下降,隨后隨著實驗時間延長,實驗組大鼠體重呈上升趨勢,但增速相比對照組降低。2硫堇染色結(jié)果對照組神經(jīng)細胞結(jié)構(gòu)清晰,尼氏體均勻分布于胞漿內(nèi)。束縛加冰水游泳實驗1天組和3天組,尼氏體無明顯變化;隨著應激時間的延長,7天組下丘腦部分神經(jīng)細胞發(fā)生水腫,尼氏體結(jié)構(gòu)不清;14天組部分神經(jīng)細胞內(nèi)尼氏體消失,神經(jīng)細胞固縮濃染,21天組與14天組相比上述病理性改變更為明顯。3fjb染色結(jié)果對照組和束縛加冰水游泳實驗1天組偶見變性壞死陽性細胞,實驗3、7天組變性壞死細胞數(shù)目增多,實驗14、21天組變性壞死細胞數(shù)目明顯增多。4elisa檢測結(jié)果與對照組相比,束縛加冰水游泳實驗組大鼠血清中糖皮質(zhì)激素水平從第3天開始顯著性增高,7天達到高峰,14天維持在較高水平,但21天時糖皮質(zhì)激素水平顯著性降低,與對照組相比無明顯差異。5腎上腺he染色結(jié)果與對照組大鼠腎上皮質(zhì)束狀帶彌漫大明細胞相比,實驗1天組未見明顯變化,3天、7天組大暗細胞增多,14和21天組大暗細胞明顯增多。6免疫組織化學染色結(jié)果6.1grp78免疫組織化學染色結(jié)果束縛加冰水游泳實驗1、3、7組的大鼠下丘腦grp78神經(jīng)細胞陽性比例與對照組相比呈明顯增高趨勢,且在實驗3天組到達高峰;實驗14天組的神經(jīng)細胞陽性比例與對照組相比沒有明顯差別;實驗21天組的神經(jīng)細胞陽性比例與對照組相比降低。6.2atf4免疫組織化學染色結(jié)果束縛加冰水游泳實驗1、3、7、14、21組的大鼠下丘腦atf4神經(jīng)細胞陽性比例與對照組相比呈明顯增高趨勢,且在7天組到達高峰,總體趨勢為先增高后略有下降。6.3atf6免疫組織化學染色結(jié)果束縛加冰水游泳實驗1天組與對照組的大鼠下丘腦atf6神經(jīng)細胞陽性比例沒有顯著區(qū)別,實驗3、7、14天組神經(jīng)細胞陽性比例呈現(xiàn)明顯升高趨勢,在14天組到達高峰,21天組回落,且神經(jīng)細胞陽性比例高于對照組。6.4jnk免疫組織化學染色結(jié)果束縛加冰水游泳實驗1、3、7、14天組大鼠jnk神經(jīng)細胞陽性比例明顯高于對照組,且呈上升趨勢并在14天到達高峰,21天組神經(jīng)細胞陽性比例略高于7天組。6.5chop免疫組織化學染色結(jié)果束縛加冰水游泳實驗1天組chop神經(jīng)細胞陽性比例與對照組相比沒有明顯差異,3、7、14、21天組神經(jīng)細胞陽性比例明顯高于對照組,且在14天到達高峰。6.6neun免疫組織化學染色結(jié)果束縛加冰水游泳實驗1、3、7天組和對照組的下丘腦所有細胞中神經(jīng)細胞所占比例相比沒有明顯區(qū)別,實驗14天和21天組神經(jīng)細胞比例明顯降低。結(jié)論:本研究成功建立了不同時程束縛加冰水游泳大鼠應激模型,采用酶聯(lián)免疫吸附試驗、硫堇染色、fluoro-jadeb染色、he染色及免疫組織化學染色進行了系列研究。實驗結(jié)果表明較長時程應激可導致下丘腦神經(jīng)細胞的損傷。內(nèi)質(zhì)網(wǎng)應激PERK-ATF4-CHOP和JNK通路被激活,ATF4、CHOP和JNK呈現(xiàn)出持續(xù)性的高表達,提示較長時程的應激導致的下丘腦神經(jīng)細胞的損傷與內(nèi)質(zhì)網(wǎng)應激PERK-ATF4-CHOP通路和JNK通路的激活及高表達有明顯相關(guān)關(guān)系。
[Abstract]:Objective: stress is a systemic nonspecific adaptive response regulated by the body through nerve, endocrinology and other systems. Moderate stress can increase the body's ability to resist external risk factors, but excessive stress can also cause injury to the body, affecting the body's normal psychological, physiological and metabolic functions, mental and psychological stress. Events can be seen everywhere in life. In forensic forensic cases, a case of a criminal suspect or a person who has suffered a major life event is often encountered by a criminal suspect or a person who has suffered a major life event. There is no obvious injury through the examination of the corpse table. It is difficult to determine the cause of death by the systematic anatomy of the pathological changes. It is found that there is a common characteristic, that is, the state of psychological stress is clear. On the premise of eliminating all the causes of death, some scholars believe that this kind of death is a stressful death, but it still lacks sufficient scientific basis, and it still needs to be supported by experimental research. The hypothalamus is an important regulation of the stress response. In this study, the pathological changes of hypothalamus, changes of protein expression in endoplasmic reticulum stress signaling pathway and changes of glucocorticoid levels in the hypothalamus were systematically observed on the basis of the successful establishment of a rat model of swimming stress in the hypothalamus. The purpose of this study was to explore whether the injury of the hypothalamic nerve cells caused by stress had endoplasmic reticulum. The participation of net stress provides a pathomorphological basis for the study of the mechanism of death under stress. Methods: 1 rats were randomly divided into 100 rats (200 + 20) g SD rats, divided into 1 days, 3 days, 7 days, 14 days and 21 days, 10 rats in each group, and set the corresponding time points in 10. At the same time, 6 hours (8:00-14:00) was shackled, and the water was fasted and the water was swallowed for 5 minutes after the end of the shackle. When the stress group was bound, the rats in the normal control group were kept in the cage, and the same fasting water was 6 hours. The rest of the time rats were free to eat and drink. The rats in each group and the corresponding control group were killed at the end of the experiment for second days. ELISA detection of blood serum and blood serum, the brain tissue was fixed, dehydrated, transparent, embedded, continuous slices and other treatments were treated with.1) to record the body weight change.2) in rats), the nelsbody changes of the nerve cells were observed by the special staining of thio.2) and the Fluoro-jade B staining was used to observe the neurodegeneration and necrosis of the nerve by Fluoro-jade B staining. The enzyme linked immunosorbent assay was used to detect the serum. The changes in the concentration of corticosteroids.5) he staining and the observation of the adrenal pathological changes.6) the ratio of.2 positive cells to the expression of ers related protein and the statistical analysis of the.2 positive cells by immunohistochemical staining: the proportion of positive cells (brown neurons) in the immunohistochemical experiment was statistically analyzed by the panoramic tissue cell quantitative analysis system. The tissue cell count has been widely accepted in foreign countries. Statistical analysis was carried out by spss21.0 software. The average number of each group was compared with single factor variance analysis (ANOVA), and the minimum significant difference (leastsignificantdifference, LSD) was used as the 22 comparison, with P0.05 as the significant difference. Results: the results of weight change of the 1 rats were with the results. In the control group, the weight of rats in the 1 day group was slightly lower than the control group, and the weight of the rats in the experimental group increased with the time of experiment. But the growth rate of the experimental group was more clear than the control group, and the Nissl body was evenly distributed in the cytoplasm of the control group with.2 thio staining. There was no obvious change in Nissl body in the 1 day group and the 3 day group. With the prolongation of the stress time, the hypothalamic neurons in the 7 day group were edema, the Nissl body structure was not clear, the Nissl body in the 14 day group disappeared, the nerve cells were condensed, and the 21 day group was more obviously.3fjb compared with the 14 day group. The positive cells in the control group and the 1 day group of 1 days of the binding and ice water swimming test, the number of denatured necrotic cells in the 3,7 day group increased, the number of the denatured necrotic cells increased significantly in the experimental group 14,21 days and the results of.4elisa detection were compared with the control group. The levels of glucocorticoid in the serum of the rats in the binding and ice water swimming test group were from third. The day began to increase significantly, the 7 days reached the peak, the 14 day maintained at a higher level, but the level of glucocorticoid decreased significantly at 21 days. Compared with the control group, there was no significant difference in the.5 adrenal HE staining results compared with the control group of the upper renal cortex of the renal cortex, the 1 day group had no significant changes, 3 days, the 7 day group of large dark cells increased. More, 14 and 21 days, the number of large dark cells was significantly increased by.6 immunohistochemical staining results of 6.1grp78 immunohistochemical staining. The positive proportion of GRP78 neurons in the hypothalamus of the 1,3,7 group was significantly higher than that of the control group, and reached the peak in the 3 day group, and the nerve cell Yang in the group of 14 days of the experiment There was no significant difference in sex ratio compared with the control group; the percentage of positive nerve cells in the 21 day group was significantly higher than that in the control group. The percentage of ATF4 neurons in the hypothalamus of the 1,3,7,14,21 group was significantly higher than that in the control group. The ratio of the positive ratio of the ATF4 to the control group was significantly higher than that in the control group, and the group reached a higher level in the 7 day group. Peak, the overall trend was increased first and then slightly decreased with.6.3atf6 immunohistochemical staining and swimming test of ice water for 1 days. There was no significant difference in the positive proportion of ATF6 neurons in the hypothalamus of the rats in the control group. The positive proportion of the nerve cells in the experimental group 3,7,14 days was obviously rising, reaching the peak in the 14 day group and the 21 day group falling down. The positive proportion of nerve cells was higher than that of the control group.6.4jnk immuno histochemical staining results. The positive ratio of JNK nerve cells in the 1,3,7,14 day group was significantly higher than that of the control group. The positive proportion of the neurons in the group was higher and reached the peak in the 14 day. The positive proportion of the nerve cells in the 21 day group was slightly higher than that of the 7 day group of.6.5chop immuno histochemical staining. Results there was no significant difference in the positive proportion of chop nerve cells between the 1 day group and the control group. The positive proportion of the nerve cells in the 3,7,14,21 day group was significantly higher than that of the control group, and the results of the peak of the 14 day peak of the.6.6neun immuno histochemical staining and the ice water swimming in the 1,3,7 day group and the control group were all fine. The proportion of neurons in the cell was not significantly different, and the proportion of nerve cells in the 14 and 21 days of the experiment was significantly reduced. Conclusion: This study successfully established the stress model of swimming rats with different time constraints plus ice water, enzyme linked immunosorbent assay, thio staining, fluoro-jadeb staining, he staining and immunohistochemical staining. A series of studies. The results show that longer duration stress can cause damage to the hypothalamic nerve cells. Endoplasmic reticulum stress PERK-ATF4-CHOP and JNK pathways are activated, ATF4, CHOP and JNK present a persistent high expression, suggesting the damage of the hypothalamic nerve cells caused by longer duration stress and the endoplasmic reticulum stress PERK-ATF4-CHOP pathway and JNK passage. There is a significant correlation between the activation of the road and the high expression of the road.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：D919.4

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