中脑导水管周围灰质星形胶质细胞在大鼠束缚浸水应激中的作用

发布时间：2018-04-22 05:24

本文选题：束缚-浸水应激 + 星形胶质细胞　；参考：《山东师范大学》2015年硕士论文

【摘要】：大鼠束缚浸水应激（RWIS）模型是刺激较强的复合应激模型。该应激模型是将大鼠四肢固定，之后浸入冷水（水温21±1℃），使其在短时间内被激怒，从而被迫接受躯体和心理双重刺激的复合应激模型。此模型可使大鼠胃肠机能紊乱，并导致其胃黏膜急性损伤，所以常用其来研究应激性胃黏膜损伤。本实验室先前的研究数据表明：大鼠在束缚浸水应激条件下，调节胃机能的神经主要是副交感神经系统（PNS）。迷走传入神经可将来自胃肠道和其他脏器的感觉信息传递至脑干，脑干孤束核（NTS）中的二级神经元将传来的信息进行整合，并可通过谷氨酸、GABA等作为神经递质将整合的信息传递至相关核团，例如臂旁核。臂旁核可经下丘脑和杏仁体传递至PAG。又有胡旺平等人的研究证明[1]，延髓头端腹外侧区（RVLM）和中脑导水管周围灰质（PAG）参与了心血管活动的调控，，当慢性应激导致PAG内NO的含量降低时，其可激活RVLM的胆碱能发挥作用,进而共同调控和维系大鼠应激性高血压。PAG不但可以接收臂旁核，下丘脑，中央杏仁核，前扣带回皮层(ACC)，脑岛（INS）以及内侧前额叶皮层（PFC）的信息传入，而且其可将接收的信息经前扣带回皮层(ACC)传递至迷走神经背核（DMV），DMV再将其发出的迷走运动信息传递至胃肠道，进而调控胃肠道的功能。ERK1与ERK2是ERK家族研究最为彻底也是表达最为广泛的分子。ERK1/2被磷酸化激活后，由细胞质转移至细胞核内，之后与cAMP反应元件结合，从而调节c-fos和Jun-B等的表达。我们的研究发现在RWIS过程中，不同脑区神经元内的c-Fos蛋白表达显著增多。而且，近年来的一些研究表明，ERK1/2信号通路与炎症、应激反应的调控关系密切，被认为是细胞信号传递通路的交汇点，那么，作为c-Fos蛋白表达的上游调控分子ERK1/2是否参与RWIS过程，起何作用，尚未见报道。那么： 1.中脑导水管周围灰质（PAG）中的星形胶质细胞是否参与RWIS所导致的胃黏膜损伤？ 2.如果星形胶质细胞参与了RWIS,那么其与神经元又存在怎样的关系？ 3.中脑导水管周围灰质（PAG）中的ERK1/2信号通道是否参与RWIS所导致的胃黏膜损伤？为了解决以上的问题，设计实验如下： 1.大鼠随机分为对照组（RWIS0h）和应激组（RWIS0.5、1、2、3、5h）其中，c-Fos蛋白的表达可作为神经元被激活的标志，胶质纤维酸性蛋白（GFAP）的表达可作为星形胶质细胞被激活的标志。本实验是通过免疫组织化学染色和蛋白质免疫印迹两种技术检测大鼠束缚-浸水应激不同时段中脑导水管周围灰质（PAG)中的c-Fos蛋白和GFAP的表达情况，从而探究PAG内神经元和星形胶质细胞是否参与了RWIS以及其应激不同时间段的变化规律。 2.将实验大鼠随机分为生理盐水对照组、L-AA给药组和ASO给药组。观察ASO给药组在抑制c-Fos蛋白的表达时，其对GFAP的表达是否有影响；观察L-AA给药组在抑制GFAP的表达时，其对c-Fos蛋白的表达是否有影响。 3.大鼠随机分为对照组（RWIS0h）和应激组（RWIS0.5、1、3、5h）通过免疫组织化学染色和蛋白质免疫印迹两种技术检测大鼠束缚-浸水应激不同时段对中脑导水管周围灰质（PAG)中p-ERK1/2（ERK1/2蛋白被激活的标志）的表达情况，从而探究ERK1/2信号通路是否参与了RWIS以及其应激不同时段的变化规律;大鼠随机分为生理盐水对照组和PD98059给药组，观察PD98059给药组在抑制p-ERK1/2蛋白的表达时，其对c-Fos蛋白、GFAP的表达及胃溃疡指数是否有影响。本实验研究结果如下： 1.束缚-浸水应激过程中，中脑导水管周围灰质（PAG）中的星形胶质细胞参与了应激所引起的胃黏膜损伤。实验结果表明，与对照组相比，在RWIS应激组中，中脑导水管周围灰质（PAG）单位面积内（0.01mm2）的c-Fos蛋白和GFAP蛋白均有增多，且差异显著，说明PAG中的神经元和星形胶质细胞参与了RWIS。 2.本实验发现注射星形胶质细胞的抑制剂L-AA之后，除星形胶质细胞的活动被抑制外，神经元c-Fos蛋白的表达量也显著减少；注射c-Fos蛋白的抑制剂ASO之后，除神经元的活动被抑制外，星形胶质细胞GFAP的表达量也显著减少，提示PAG内神经元和星形胶质细胞之间可能存在着交互关系。 3.束缚-浸水应激过程中，中脑导水管周围灰质（PAG）中的星形胶质细胞参与了应激所引起的胃黏膜损伤。实验结果表明，与对照组相比，在RWIS应激组中，观察到大鼠PAG内p-ERK1/2表达量显著增多，ERK1/2蛋白的特异性抑制剂PD98059能显著抑制c-Fos蛋白和GFAP的表达，说明PAG内ERK1/2蛋白信号通路也参与了束缚-浸水应激。
[Abstract]:The rat model of restraint and immersion stress (RWIS) model is a strong compound stress model. The stress model is fixed in rats, then immerse in cold water (water temperature 21 + 1 c), so that it is irritated in a short time, which is forced to accept the compound stress model of physical and psychological stimulation. This model can cause the gastrointestinal dysfunction in rats and lead to it. Acute gastric mucosal injury, so it is commonly used to study stress gastric mucosal injury. Previous research in our laboratory shows that the main nerve regulating gastric function in rats is the parasympathetic nervous system (PNS) under restraint stress. The vagal afferent nerve can transfer sensory information from the gastrointestinal and other organs to the brain stem and brain. The two stage neurons in the nucleus of the dry solitary nucleus (NTS) integrate information and transmit integrated information as neurotransmitters as neurotransmitters, such as the paralnial nucleus, through the hypothalamus and amygdala, through the hypothalamus and amygdala to PAG. and Hu Wangping and others to prove [1], RVLM and middle medulla oblongata. The periaqueductal gray matter (PAG) participates in the regulation of cardiovascular activity. When chronic stress causes the decrease in the content of NO in PAG, it activates the effect of the cholinergic activity of the RVLM, and then co regulates and maintains the rat stress hypertension.PAG not only to receive the paracert nucleus, the hypothalamus, the central amygdala, the anterior cingulate cortex (ACC), and the insula (INS). And the information of the medial prefrontal cortex (PFC) is transmitted, and it can transmit the received information through the anterior cingulate cortex (ACC) to the dorsum of the vagus nerve (DMV). DMV then transfers the information from the vagus movement to the gastrointestinal tract, and then regulates the function of the gastrointestinal tract,.ERK1 and ERK2, which are the most thorough and most widely expressed molecular.E in the ERK family. When RK1/2 is activated by phosphorylation, it is transferred from cytoplasm to the nucleus, and then combined with the cAMP reaction element to regulate the expression of c-fos and Jun-B. Our study found that the expression of c-Fos protein in the neurons of different brain regions increased significantly during the RWIS process. Moreover, some recent studies have shown that the ERK1/2 signaling pathway and inflammation and stress are stressed. The regulation of reaction is closely related, and it is considered to be the intersection point of cell signaling pathway. Then, as the upstream regulatory molecule ERK1/2 expressed by c-Fos protein is involved in the RWIS process, it has not yet been reported.
1. are astrocytes in the periaqueductal gray (PAG) involved in gastric mucosal injury caused by RWIS?
2. if astrocytes are involved in RWIS, what is the relationship between them and neurons?
3. is the ERK1/2 signaling pathway in the periaqueductal gray (PAG) involved in gastric mucosal injury caused by RWIS?
In order to solve the above problems, the design experiment is as follows:
The 1. rats were randomly divided into the control group (RWIS0h) and the stress group (RWIS0.5,1,2,3,5h). The expression of c-Fos protein could be used as a marker for activation of neurons. The expression of glial fibrillary acidic protein (GFAP) could be used as a sign of activation of astrocytes. This experiment was conducted by two techniques of immunostaining and protein immunoblotting. The expression of c-Fos protein and GFAP in the periaqueductal gray (PAG) in different periods of restraint stress in rats was measured to explore whether the neurons in PAG and astrocytes were involved in RWIS and the change of different time periods of stress.
2. the experimental rats were randomly divided into the normal saline control group, the L-AA administration group and the ASO administration group. The effect of the ASO administration group on the expression of c-Fos protein in the expression of GFAP was observed, and the effect of the L-AA administration group on the expression of c-Fos protein in the inhibition of the expression of GFAP was observed.
The 3. rats were randomly divided into the control group (RWIS0h) and the stress group (RWIS0.5,1,3,5h) to detect the expression of the p-ERK1/2 (ERK1/2 protein stimulated) in the periaqueductal gray matter (PAG) in the mesencephalic periaqueductal gray (PAG) by immunohistochemical staining and protein immunoblotting, and to explore the ERK1/2 signaling pathway. Whether the rats were involved in RWIS and the variation of their stress different segments; rats were randomly divided into the saline control group and the PD98059 administration group. The effect of the PD98059 administration group on the expression of p-ERK1/2 protein, the expression of GFAP and the index of gastric ulcer was observed.
The results of this study are as follows:
1. the astrocytes in the mesencephalic periaqueductal gray matter (PAG) were involved in the gastric mucosal damage caused by stress during the process of binding immersion stress. The results showed that in the RWIS stress group, the c-Fos and GFAP protein in the PAG per unit area of the mesencephalic periaqueductal gray matter (0.01mm2) increased, and the difference was significant. It shows that neurons and astrocytes in PAG are involved in RWIS..
2. we found that after the inhibition of astroglia, the activity of astrocytes was suppressed, the expression of c-Fos protein was significantly reduced, and the expression of astrocyte GFAP was decreased significantly after the inhibition of the activity of c-Fos protein, ASO, suggesting the internal PAG nerve. There may be an interaction between astrocytes and astrocytes.
3. the astrocytes in the mesencephalic periaqueductal gray matter (PAG) were involved in the gastric mucosal injury caused by stress during the binding immersion stress. The experimental results showed that compared with the control group, the expression of p-ERK1/2 in the rat PAG was significantly increased in the RWIS stress group, and the specific inhibitor PD98059 of the ERK1/2 protein could significantly inhibit the c-Fos. The expression of protein and GFAP indicates that ERK1/2 signaling pathway in PAG is also involved in restraint water immersion stress.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.91

【参考文献】