内质网应激参与应激大鼠下丘脑神经细胞损伤

发布时间：2018-06-12 01:13

本文选题：下丘脑 + 束缚　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:应激是机体通过神经、内分泌以及其他系统共同参与调节的一种全身非特异适应性反应。适度的应激可以增加机体抵抗外界危险因素的能力,但过度应激也可造成机体的损伤,影响机体正常的心理、生理及代谢功能。精神、心理应激事件在生活中随处可见,在法医学检案中时常遇到被羁押的犯罪嫌疑人或遭受重大生活事件的当事人发生快速意外死亡的案例,经尸表检验无明显损伤,经系统解剖无特异性病理改变,因此死因难以确定。认真分析上述案件的背景不难发现存在一个共同的特点,即均存在明确的心理应激状况。通过系统解剖在排除所有死因的前提下,目前部分学者认为这类死亡为应激性死亡,但尚缺乏足够的科学依据,还有待于进行实验研究给予支持。下丘脑是应激反应的重要调节中枢,因此本研究在成功建立不同时程束缚加冰水游泳应激大鼠模型的基础上,系统观察了下丘脑组织病理学改变、内质网应激信号通路相关蛋白表达变化及糖皮质激素水平变化,旨在探讨应激导致的下丘脑神经细胞损伤是否有内质网应激的参与,为应激状态下死亡机制的研究提供病理形态学依据。方法:1本实验将100只体重为(200±20)g SD雄性大鼠随机分组,分为1天、3天、7天、14天和21天组,每组大鼠10只,并设定相应时间点的各正常对照组10只。采用将大鼠仰卧位固定每日6小时(8:00-14:00)的束缚处理,同时禁食水,束缚结束后进行5分钟冰水游泳。应激各组进行束缚处理时,正常对照组大鼠于饲养笼中,同样禁食水6小时,其余时间各组大鼠自由进食、饮水。各实验组及对应对照组大鼠于实验结束第二天处死,取血留血清做ELISA检测,脑组织经固定、脱水、透明、包埋、连续切片等处理后待做各种染色使用。1)记录大鼠体重变化。2)硫堇特殊染色观察神经细胞尼氏体变化。3)Fluoro-jade B染色观察神经细胞变性坏死情况。4)酶联免疫吸附试验检测血清中糖皮质激素浓度变化。5)he染色观察肾上腺病理学改变。6)免疫组织化学染色观察ers相关蛋白表达变化。2阳性细胞比例及统计学分析:运用全景组织细胞定量分析系统对免疫组化实验中阳性细胞(棕色神经细胞)比例进行统计,目前此软件用于组织细胞计数已在国外得到广泛认可。采用spss21.0统计软件进行统计学分析,各组均数的比较行单因素方差分析(anova),用最小显著差法(leastsignificantdifference,lsd)作两两比较,以p0.05为有显著统计学差异。结果:1大鼠体重变化结果随时间的延长,对照组大鼠体重呈上升趋势。与对照组相比,实验1天组大鼠体重略有下降,随后随着实验时间延长,实验组大鼠体重呈上升趋势,但增速相比对照组降低。2硫堇染色结果对照组神经细胞结构清晰,尼氏体均匀分布于胞浆内。束缚加冰水游泳实验1天组和3天组,尼氏体无明显变化;随着应激时间的延长,7天组下丘脑部分神经细胞发生水肿,尼氏体结构不清;14天组部分神经细胞内尼氏体消失,神经细胞固缩浓染,21天组与14天组相比上述病理性改变更为明显。3fjb染色结果对照组和束缚加冰水游泳实验1天组偶见变性坏死阳性细胞,实验3、7天组变性坏死细胞数目增多,实验14、21天组变性坏死细胞数目明显增多。4elisa检测结果与对照组相比,束缚加冰水游泳实验组大鼠血清中糖皮质激素水平从第3天开始显著性增高,7天达到高峰,14天维持在较高水平,但21天时糖皮质激素水平显著性降低,与对照组相比无明显差异。5肾上腺he染色结果与对照组大鼠肾上皮质束状带弥漫大明细胞相比,实验1天组未见明显变化,3天、7天组大暗细胞增多,14和21天组大暗细胞明显增多。6免疫组织化学染色结果6.1grp78免疫组织化学染色结果束缚加冰水游泳实验1、3、7组的大鼠下丘脑grp78神经细胞阳性比例与对照组相比呈明显增高趋势,且在实验3天组到达高峰;实验14天组的神经细胞阳性比例与对照组相比没有明显差别;实验21天组的神经细胞阳性比例与对照组相比降低。6.2atf4免疫组织化学染色结果束缚加冰水游泳实验1、3、7、14、21组的大鼠下丘脑atf4神经细胞阳性比例与对照组相比呈明显增高趋势,且在7天组到达高峰,总体趋势为先增高后略有下降。6.3atf6免疫组织化学染色结果束缚加冰水游泳实验1天组与对照组的大鼠下丘脑atf6神经细胞阳性比例没有显著区别,实验3、7、14天组神经细胞阳性比例呈现明显升高趋势,在14天组到达高峰,21天组回落,且神经细胞阳性比例高于对照组。6.4jnk免疫组织化学染色结果束缚加冰水游泳实验1、3、7、14天组大鼠jnk神经细胞阳性比例明显高于对照组,且呈上升趋势并在14天到达高峰,21天组神经细胞阳性比例略高于7天组。6.5chop免疫组织化学染色结果束缚加冰水游泳实验1天组chop神经细胞阳性比例与对照组相比没有明显差异,3、7、14、21天组神经细胞阳性比例明显高于对照组,且在14天到达高峰。6.6neun免疫组织化学染色结果束缚加冰水游泳实验1、3、7天组和对照组的下丘脑所有细胞中神经细胞所占比例相比没有明显区别,实验14天和21天组神经细胞比例明显降低。结论:本研究成功建立了不同时程束缚加冰水游泳大鼠应激模型,采用酶联免疫吸附试验、硫堇染色、fluoro-jadeb染色、he染色及免疫组织化学染色进行了系列研究。实验结果表明较长时程应激可导致下丘脑神经细胞的损伤。内质网应激PERK-ATF4-CHOP和JNK通路被激活,ATF4、CHOP和JNK呈现出持续性的高表达,提示较长时程的应激导致的下丘脑神经细胞的损伤与内质网应激PERK-ATF4-CHOP通路和JNK通路的激活及高表达有明显相关关系。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：D919.4

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