本文選題：穹窿切斷術(shù) + MR��； 參考：《中國醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的 下丘腦—垂體—腎上腺(hypothalamic-pituitary-adrenal，HPA)軸作為神經(jīng)內(nèi)分泌免疫網(wǎng)絡(luò)的樞紐，在機體內(nèi)環(huán)境平衡維持及機體衰老過程的調(diào)節(jié)中起著十分重要的作用。糖皮質(zhì)激素作用于垂體和下丘腦構(gòu)成HPA軸負反饋調(diào)節(jié)的主要通路，然而現(xiàn)有大量資料表明，下丘腦以上結(jié)構(gòu)特別是海馬參與了HPA軸的調(diào)節(jié)，而且是具有重要意義的負反饋調(diào)節(jié)。 目前的研究普遍認(rèn)為海馬通過赫皮質(zhì)激素受體(Mineralocorticoid Receptor，MR)和糖皮質(zhì)激素受體(Glucocorticoid Receptor，GR)對下丘腦的室旁核產(chǎn)生了抑制作用。靜息條件下，糖皮質(zhì)激素與海馬中的MR結(jié)合參與基礎(chǔ)水平的HPA軸負反饋調(diào)節(jié)；應(yīng)激條件下，高水平的應(yīng)激激素與海馬中的GR結(jié)合，抑制HPA軸的過度反應(yīng)或使應(yīng)激狀態(tài)的HPA軸功能恢復(fù)到基礎(chǔ)水平。MR、GR這兩個受體屬于核受體家族，核受體是能與特異性激素結(jié)合的蛋白，主要是一類配基依賴性轉(zhuǎn)錄調(diào)節(jié)因子，通過基因表達誘導(dǎo)或者抑制細胞的增殖，分化和死忙，導(dǎo)致多種生物效應(yīng)。但是從海馬到室旁核沒有直接的神經(jīng)投射，間接的路徑都要經(jīng)過穹窿�，F(xiàn)有研究報道穹窿切斷也可以影響室旁核的CRH和AVP的水平，由此引發(fā)我們推測海馬對下丘腦室旁核的抑制作用是否媒介了穹窿。 本實驗建立大鼠穹窿切斷模型，采用免疫組織化學(xué)、western blotting、圖像分析等方法對穹窿切斷后大鼠海馬神經(jīng)元MR、GR的表達進行了研究，希望為海馬神經(jīng)元MR、GR調(diào)節(jié)HPA軸的相關(guān)機制提供實驗依據(jù)。 實驗方法 一、實驗動物及分組 成年健康雄性Wistar大鼠90只，體重200—220g，中國醫(yī)科大學(xué)實驗動物中心提供，，實驗室條件飼養(yǎng)。隨機分為正常對照組(10只)、穹窿切斷組(分為0天、4天、7天、10天組，每組10只)、假手術(shù)組(未切穹窿，分為0天、4天、7天、10天組，每組10只) 二、動物模型的建立 將大鼠用戊巴比妥將其麻醉后，固定于腦立體定位儀(江灣IC型)，常規(guī)消毒、剪毛，正中切開頭皮，暴露出顱骨，于前囟后1.5mm，中線外左右1.0mm處，用電動開顱器鑿開顱骨，切開硬腦膜，用自制的雙刃刀先置于上述部位的腦表面，(相當(dāng)于穹窿部位)，續(xù)降刀6mm，切斷穹窿，降刀時間約1分鐘，假手術(shù)組降刀2mm，(未切穹窿)，刀在腦中均停留5分鐘。清潔顱面，縫合頭皮，常規(guī)飼養(yǎng)，抗菌素控制感染。 三、大鼠穹窿切斷模型的鑒定 對正常組和穹窿切斷組大鼠進行海馬膽堿能纖維AchE組織化學(xué)染色。 四、免疫組織化學(xué)染色 各組大鼠經(jīng)4％多聚甲醛灌流固定、取材，制作冰凍切片進行免疫組化SABC法染色，(MR工作濃度為1：200、GR工作濃度為1：500)，DAB顯色，中性樹膠封片，光學(xué)顯微鏡(OLYMPUS，BX60，Japan)下觀察，攝片。并作圖像分析。 五、Western Blotting 取新鮮海馬組織經(jīng)勻漿、超聲粉碎后高速低溫離心，提取上清蛋白，電泳，轉(zhuǎn)膜，經(jīng)封閉后加一抗MR、GR(MR工作濃度1：500，GR工作濃度1：1000)4℃過夜；HRP標(biāo)記IgG(工作濃度1：1500)孵育2h，DAB法顯色，掃描條帶計算灰度值。 實驗結(jié)果 一、大鼠海馬纖維AchE染色結(jié)果 正常海馬結(jié)構(gòu)內(nèi)有豐富的膽堿能纖維，纖維粗細均勻且交織成網(wǎng)。穹窿切斷組AchE陽性纖維大量喪失，觀察海馬CA1區(qū)神經(jīng)纖維，纖維變細排列紊亂且密度降低。說明穹窿切斷模型建立成功。 二、NR、GR免疫組化反應(yīng)染色結(jié)果 正常組MR、GR免疫反應(yīng)陽性產(chǎn)物呈棕黃色表達，位于細胞質(zhì)和細胞核內(nèi)，MR主要位于海馬CA1、CA2、CA3區(qū)的錐體細胞層和齒狀回的顆粒細胞層的神經(jīng)元。GR主要位于海馬CA1、CA2區(qū)的錐體細胞層和齒狀回的顆粒細胞層的神經(jīng)元。觀察海馬CA1區(qū)，MR、GR表達陽性的錐體細胞數(shù)目較多，染色較深，細胞排列較整齊。各假手術(shù)組MR、GR的表達與正常組相似，0、4天穹窿切斷組與正常組、相應(yīng)假手術(shù)組相比MR、GR免疫反應(yīng)陽性細胞的數(shù)量與形態(tài)沒有明顯改變，7、10天穹窿切斷組海馬CA1區(qū)MR、GR免疫反應(yīng)陽性細胞數(shù)較少，細胞形態(tài)變小，染色淺。而且10天穹窿切斷組較7天穹窿切斷組免疫反應(yīng)陽性細胞數(shù)少，染色更淺。 三、MR、GR Western BIotting結(jié)果 Western Blotting檢測發(fā)現(xiàn)海馬MR、GR的表達在7天、10天穹窿切斷組較正常組、假手術(shù)組減少，且10天組較7天組表達量少。0、4天組與正常組、假手術(shù)組相比無明顯差異。各假手術(shù)組MR、GR的表達與正常組相比無明顯差異。 四、圖像分析結(jié)果 7天、10天穹窿切斷組，海馬神經(jīng)元MR、GR表達較正常組、假手術(shù)組表達量減少(p＜0.05)，10天較7天組表達量減少(p＜0.05)0、4天穹窿切斷組與正常組、假手術(shù)組相比無明顯差異(p＞0.05)。各假手術(shù)組MR、GR的表達與正常組相比無明顯差異(p＞0.05)。 結(jié)論 穹窿切斷后，大鼠海馬神經(jīng)元MR、GR表達減弱，提示可能穹窿切斷后海馬對HPA軸的抑制作用減弱。
[Abstract]:objective
The hypothalamus pituitary adrenal (hypothalamic-pituitary-adrenal, HPA) axis, as the hub of the neuroendocrine immune network, plays a very important role in the maintenance of environmental balance and the regulation of the aging process in the body. Glucocorticoid acts as the main pathway of HPA axis negative feedback regulation in the pituitary and hypothalamus, but now it is the main pathway of the negative feedback regulation of the pituitary and hypothalamus. A large number of data indicate that the hypothalamic structure, especially the hippocampus, is involved in the regulation of the HPA axis, and is a significant negative feedback regulation.
At present, it is widely believed that the hippocampus inhibits the paraventricular nucleus of the hypothalamus through the Mineralocorticoid Receptor (MR) and Glucocorticoid Receptor (GR). In resting conditions, the glucocorticoid and the MR in the hippocampus are involved in the negative feedback regulation of the HPA axis of the basal level; the stress bar is a stress bar. A high level of stress hormone combined with GR in the hippocampus to inhibit the overreaction of the HPA axis or to restore the HPA axis function of the stress state to the basic level.MR. The two receptors of GR belong to the nuclear receptor family, and the nuclear receptor is a protein binding to the specific hormone, mainly a class of ligand dependent transcription factors, which are induced by gene expression. The proliferation, differentiation, and death of cells lead to a variety of biological effects. But there is no direct neural projection from the hippocampus to the paraventricular nucleus and the indirect path passes through the fornix. The present study reports that the fornix cut can also affect the level of CRH and AVP in the paraventricular nucleus, which leads us to speculate on the inhibition of the hippocampus to the paraventricular nucleus of the inferior colliculus. Whether the action mediate the fornix.
In this experiment, rat fornix cutting model was established. Immunohistochemistry, Western blotting and image analysis were used to study the expression of MR and GR in hippocampal neurons of rats after fornix fornix cutting. It was hoped to provide experimental evidence for the mechanism of MR and GR to regulate the HPA axis of hippocampal neurons.
Experimental method
First, experimental animals and groups
90 adult healthy male Wistar rats, weighing 200 - 220g, were provided by laboratory animal center of China Medical University and kept in laboratory. They were randomly divided into normal control group (10 rats), fornix cutting group (divided into 0 days, 4 days, 7 days, 10 days, 10 in each group), and the sham operation group (0 days, 4 days, 7 days, 10 days, 10 only)
Two, the establishment of animal model
The rats were anaesthetized with pentobarbital, fixed to the brain stereotaxis (Jiangwan type IC), routinely sterilizing, cutting hair, cutting the scalp in the midline, exposing the skull, 1.5mm in the anterior fontanel, and outside the middle line 1.0mm. The skull was cut open and the dura was cut with an electric craniator, and the surface of the brain was first placed with a self-made double-edged knife (equivalent to the fornix). 6mm, cut the fornix, cut the fornix, cut the knife time about 1 minutes, the sham operation group reduced the knife 2mm, (not cut the fornix), the knife stayed in the brain for 5 minutes. Clean the craniofacial, suture the scalp, the routine feeding, antibiotics control infection.
Three, identification of fornix fornix model in rats
The hippocampal cholinergic fibers of rats in the normal group and fornix transection group were stained with AchE histochemistry.
Four, immunohistochemical staining
The rats were perfused and fixed by 4% polyformaldehyde in each group. The frozen sections were made by immunohistochemical SABC staining. (MR working concentration was 1:200, GR working concentration was 1:500), DAB color, neutral gum seal, optical microscopy (OLYMPUS, BX60, Japan) were observed and photographed, and image analysis was made.
Five, Western Blotting
The fresh hippocampal tissues were homogenized and centrifuged at high speed and low temperature after ultrasonic comminution. The supernatant protein, electrophoresis, and membrane were extracted. After closed, a anti MR, GR (MR working concentration 1:500, GR working concentration 1:1000) were 4 degrees centigrade; HRP labeled IgG (working concentration 1:1500) was incubated for 2H, DAB method was coloured, and the scanning strip was used to calculate gray value.
experimental result
The results of AchE staining in the hippocampus of rats
There were plenty of cholinergic fibers in the normal hippocampal structure. The fibrous thickness was uniform and interwoven into the net. The AchE positive fibers in the fornix fornix group were lost and the nerve fibers in the CA1 region of the hippocampus were observed. The fibers were arranged in a thin arrangement and the density was reduced.
Two, NR, GR immunohistochemical staining results
In the normal group MR, the positive products of the GR immunoreaction were brown and yellow, located in the cytoplasm and nucleus, and MR was mainly located in the hippocampus CA1. The pyramidal layer of the CA2, CA3 region and the granular cell layer of the dentate gyrus were mainly located in the hippocampal CA1, the pyramidal layer of the CA2 region and the granular cell layer of the dentate gyrus. The CA1 region of the hippocampus, MR, GR was observed. The number of positive pyramidal cells was more, the coloring was deeper and the cells were arranged neatly. The expression of MR and GR in the sham operation group was similar to that of the normal group. The number and the morphology of the 0,4 vault cut group were compared with the normal group and the corresponding sham operation group, compared with the corresponding sham operation group, the number and shape of the GR immunoreactive cells were not changed obviously. The CA1 region MR of the hippocampus of 7,10 days fornix fornix group and the GR immune response were found. The number of positive cells was smaller, cell morphology was smaller and staining was shallow. Moreover, the number of immunoreactive cells in the fornix cut group was less than that in the 7 day fornix transection group, and the staining was more shallow on the 10 day.
Three, MR, GR Western BIotting results
Western Blotting detected the expression of MR in the hippocampus, the expression of GR in 7 days, the 10 day of the fornix cutting group was less than the normal group, the sham operation group decreased, and the 10 day group compared with the 7 day group was less.0,4 days than the normal group, and there was no significant difference between the sham operation group and the sham operation group. The expression of GR in the sham operation group was not significantly different from that of the normal group.
Four, image analysis results
On the 7 day, the 10 day fornix cutting group, the hippocampal neurons MR, GR expression was more than the normal group, the sham operation group decreased (P < 0.05), the 10 day group was less than the 7 day group (P < 0.05) 0,4 dome cut group and the normal group, the sham operation group had no significant difference (P > 0.05). The expression of MR in the artificial hand group was not significantly different from that of the normal group (P > 0.05).
conclusion
After fornix transection, the expression of MR and GR in hippocampal neurons of rats was weakened, suggesting that the inhibitory effect of hippocampus on HPA axis might be weakened after fornix transection.
【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】：R322.811

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