本文選題：束縛-浸水應激 + 星形膠質細胞��； 參考：《山東師范大學》2015年碩士論文

【摘要】：大鼠束縛浸水應激（RWIS）模型是刺激較強的復合應激模型。該應激模型是將大鼠四肢固定，之后浸入冷水（水溫21±1℃），使其在短時間內被激怒，從而被迫接受軀體和心理雙重刺激的復合應激模型。此模型可使大鼠胃腸機能紊亂，并導致其胃黏膜急性損傷，所以常用其來研究應激性胃黏膜損傷。本實驗室先前的研究數(shù)據(jù)表明：大鼠在束縛浸水應激條件下，調節(jié)胃機能的神經主要是副交感神經系統(tǒng)（PNS）。迷走傳入神經可將來自胃腸道和其他臟器的感覺信息傳遞至腦干，腦干孤束核（NTS）中的二級神經元將傳來的信息進行整合，并可通過谷氨酸、GABA等作為神經遞質將整合的信息傳遞至相關核團，例如臂旁核。臂旁核可經下丘腦和杏仁體傳遞至PAG。又有胡旺平等人的研究證明[1]，延髓頭端腹外側區(qū)（RVLM）和中腦導水管周圍灰質（PAG）參與了心血管活動的調控，，當慢性應激導致PAG內NO的含量降低時，其可激活RVLM的膽堿能發(fā)揮作用,進而共同調控和維系大鼠應激性高血壓。PAG不但可以接收臂旁核，下丘腦，中央杏仁核，前扣帶回皮層(ACC)，腦島（INS）以及內側前額葉皮層（PFC）的信息傳入，而且其可將接收的信息經前扣帶回皮層(ACC)傳遞至迷走神經背核（DMV），DMV再將其發(fā)出的迷走運動信息傳遞至胃腸道，進而調控胃腸道的功能。ERK1與ERK2是ERK家族研究最為徹底也是表達最為廣泛的分子。ERK1/2被磷酸化激活后，由細胞質轉移至細胞核內，之后與cAMP反應元件結合，從而調節(jié)c-fos和Jun-B等的表達。我們的研究發(fā)現(xiàn)在RWIS過程中，不同腦區(qū)神經元內的c-Fos蛋白表達顯著增多。而且，近年來的一些研究表明，ERK1/2信號通路與炎癥、應激反應的調控關系密切，被認為是細胞信號傳遞通路的交匯點，那么，作為c-Fos蛋白表達的上游調控分子ERK1/2是否參與RWIS過程，起何作用，尚未見報道。那么： 1.中腦導水管周圍灰質（PAG）中的星形膠質細胞是否參與RWIS所導致的胃黏膜損傷？ 2.如果星形膠質細胞參與了RWIS,那么其與神經元又存在怎樣的關系？ 3.中腦導水管周圍灰質（PAG）中的ERK1/2信號通道是否參與RWIS所導致的胃黏膜損傷？ 為了解決以上的問題，設計實驗如下： 1.大鼠隨機分為對照組（RWIS0h）和應激組（RWIS0.5、1、2、3、5h）其中，c-Fos蛋白的表達可作為神經元被激活的標志，膠質纖維酸性蛋白（GFAP）的表達可作為星形膠質細胞被激活的標志。本實驗是通過免疫組織化學染色和蛋白質免疫印跡兩種技術檢測大鼠束縛-浸水應激不同時段中腦導水管周圍灰質（PAG)中的c-Fos蛋白和GFAP的表達情況，從而探究PAG內神經元和星形膠質細胞是否參與了RWIS以及其應激不同時間段的變化規(guī)律。 2.將實驗大鼠隨機分為生理鹽水對照組、L-AA給藥組和ASO給藥組。觀察ASO給藥組在抑制c-Fos蛋白的表達時，其對GFAP的表達是否有影響；觀察L-AA給藥組在抑制GFAP的表達時，其對c-Fos蛋白的表達是否有影響。 3.大鼠隨機分為對照組（RWIS0h）和應激組（RWIS0.5、1、3、5h）通過免疫組織化學染色和蛋白質免疫印跡兩種技術檢測大鼠束縛-浸水應激不同時段對中腦導水管周圍灰質（PAG)中p-ERK1/2（ERK1/2蛋白被激活的標志）的表達情況，從而探究ERK1/2信號通路是否參與了RWIS以及其應激不同時段的變化規(guī)律;大鼠隨機分為生理鹽水對照組和PD98059給藥組，觀察PD98059給藥組在抑制p-ERK1/2蛋白的表達時，其對c-Fos蛋白、GFAP的表達及胃潰瘍指數(shù)是否有影響。 本實驗研究結果如下： 1.束縛-浸水應激過程中，中腦導水管周圍灰質（PAG）中的星形膠質細胞參與了應激所引起的胃黏膜損傷。實驗結果表明，與對照組相比，在RWIS應激組中，中腦導水管周圍灰質（PAG）單位面積內（0.01mm2）的c-Fos蛋白和GFAP蛋白均有增多，且差異顯著，說明PAG中的神經元和星形膠質細胞參與了RWIS。 2.本實驗發(fā)現(xiàn)注射星形膠質細胞的抑制劑L-AA之后，除星形膠質細胞的活動被抑制外，神經元c-Fos蛋白的表達量也顯著減少；注射c-Fos蛋白的抑制劑ASO之后，除神經元的活動被抑制外，星形膠質細胞GFAP的表達量也顯著減少，提示PAG內神經元和星形膠質細胞之間可能存在著交互關系。 3.束縛-浸水應激過程中，中腦導水管周圍灰質（PAG）中的星形膠質細胞參與了應激所引起的胃黏膜損傷。實驗結果表明，與對照組相比，在RWIS應激組中，觀察到大鼠PAG內p-ERK1/2表達量顯著增多，ERK1/2蛋白的特異性抑制劑PD98059能顯著抑制c-Fos蛋白和GFAP的表達，說明PAG內ERK1/2蛋白信號通路也參與了束縛-浸水應激。
[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.

【學位授予單位】：山東師范大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.91

【參考文獻】

相關期刊論文 前10條

1 張輝,段麗,饒志仁;大鼠腦干星形膠質細胞和神經元對一側脛腓骨骨折后的即刻應激反應及其相互關系[J];第四軍醫(yī)大學學報;2002年05期

2 盧佩彥;李澤鴻;郭勇;競花蘭;林文盈;劉小山;;海洛因對神經元原癌基因c-fos表達的抑制作用[J];法醫(yī)學雜志;2010年05期

3 李小媚;李愛萍;;小膠質細胞的發(fā)育和功能[J];解剖學研究;2010年03期

4 笪紅遠;;應激性胃潰瘍實驗研究及中藥防治作用研究進展[J];中國臨床藥理學雜志;2005年06期

5 陳建勇;王聰;王娟;曹禮榮;;MAPK信號通路研究進展[J];中國醫(yī)藥科學;2011年08期

6 槐瑞托,牛麗靜,管振龍;大鼠中腦導水管周圍灰質的分區(qū)及其細胞構筑特點[J];河北師范大學學報;2002年04期

7 劉俊華;趙正卿;雷輝;段麗;曹榮;萬琪;趙鋼;饒志仁;;熱應激對大鼠腦橋臂旁核Fos和GFAP表達的影響[J];神經解剖學雜志;2007年04期

8 楊曉運,李智,秦綠葉,于常海;星形膠質細胞和神經元之間谷氨酸-谷氨酰胺的代謝偶聯(lián)[J];生理科學進展;2003年04期

9 李金星;趙海梅;李玉;王

本文編號：1785799