【摘要】：目的：通過建立急性CO中毒遲發(fā)型腦�。―EACMP）大鼠模型，觀察HIF-1α及EPO在相應動物模型中的表達變化，探討HIF-1α及EPO在DEACMP發(fā)病機制中的作用。方法：78只健康雄性成年SD大鼠（SPF級），體重在240～280g之間，隨機分為三組：空白對照組（BC組）18只、空氣對照組（AC組）30只、急性一氧化碳中毒遲發(fā)性腦病組（CO組）30只。其中CO組采用腹腔注射法建立DEACMP大鼠模型，AC組采用腹腔注射法注射等量空氣，BC組不進行任何手術操作。按中毒后1d、3d、7d、14d、21d、28d六個時間點將三個組各分為6個亞組，其中CO組和AC組每個亞組5只大鼠，BC組每個亞組3只大鼠。觀察大鼠急性CO中毒表現和大鼠動脈血COHb濃度及中毒后期運用Morris水迷宮實驗檢測大鼠認知行為學改變。各組大鼠的腦組織切片均應用HE染色觀察大鼠海馬區(qū)病理變化，用TUNEL染色檢測海馬區(qū)錐體細胞凋亡情況，應用免疫組化染色檢測海馬區(qū)HIF-1α、EPO蛋白的表達。結果：1、Morris水迷宮實驗結果：CO組大鼠在染毒后出現平均逃避潛伏期延長，且在染毒后14d、21d及28d時，CO組較BC組和AC組比較差異均有顯著統計學意義（P0.01)。染毒后CO組大鼠在第Ⅳ象限運動時間有縮短趨勢，且染毒14d及染毒28d時，CO組較BC組及AC組比較差異有統計學意義（P0.05)。染毒后CO組大鼠穿越平臺次數有降低趨勢，染毒28d時，CO組穿越平臺次數較其余兩組低，差異有統計學意義（P0.05）。2、HE染色：CO組大鼠在急性CO中毒后出現海馬區(qū)錐體細胞數目減少、細胞排列松散、細胞水腫、核固縮、核碎裂等病理學改變。3、TUNEL法檢測：CO組大鼠海馬區(qū)錐體細胞于染毒3d時凋亡開始增多，，7d時達到高峰，28d時仍有少量細胞凋亡，各時間點凋亡指數與BC組及AC組比較，差異具有顯著性（P0.01)。4、免疫組化染色結果：1）HIF-1α蛋白測定：HIF-1α在BC組及AC組中表達較少，且各時間點間差異不具有顯著性，CO組在染毒1d時出現HIF-1α的表達升高，3d達到峰值，28d時仍有表達，表達量明顯高于BC組及AC組，差異具有顯著性（P0.01）；2）EPO蛋白測定：在BC組及AC組中有少量表達，且各時間點間差異不具有顯著性，CO組在染毒1d時明顯升高，3d為高峰，7d時下降，染毒1d、3d、7d、14d時CO組較BC組與AC組差異具有顯著性（P0.01）。結論：1.腹腔注射CO法制作DEACMP大鼠模型，操作簡單，是研究DEACMP較為理想的動物模型；2.Morris水迷宮實驗是初步判斷大鼠DEACMP較為可靠的依據；3.HIF-1α可能通過誘導急性CO中毒后海馬區(qū)神經元的遲發(fā)性凋亡而參與DEACMP的發(fā)�。�4.EPO在急性CO中毒后中后期神經保護作用減弱，可能是導致DEACMP發(fā)病原因之一。
[Abstract]:Objective: to investigate the role of HIF-1 偽 and EPO in the pathogenesis of DEACMP by establishing a rat model of delayed encephalopathy (DEACMP) caused by acute CO poisoning, and observing the expression of HIF-1 偽 and EPO in corresponding animal models. Methods Seventy-eight healthy male adult SD rats (SPF grade), weighing between 240 and 280 g, were randomly divided into three groups: blank control group (BC group, n = 18), air control group (AC group, n = 30) and acute carbon monoxide poisoning delayed encephalopathy group (CO group, n = 30). In the CO group, the DEACMP rat model was established by intraperitoneal injection. The AC group was injected the same amount of air by intraperitoneal injection without any operation. The three groups were divided into 6 subgroups according to the six time points 1 day, 3 days, 7 days, 14 days and 21 days, 28 days after poisoning. Five rats in each subgroup of CO group and AC group were divided into 3 rats in each subgroup of BC group. The manifestations of acute CO poisoning and the concentration of COHb in arterial blood of rats were observed. Morris water maze test was used to detect the changes of cognitive behavior in rats. The pathological changes of hippocampus were observed by HE staining, apoptosis of pyramidal cells in hippocampus were detected by Tunel staining, and HIF-1 偽 EPO protein expression in hippocampus was detected by immunohistochemical staining. Results in the water maze experiment, the escape latency of the rats in the control group was longer than that in the BC group and AC group (P0.01), and there was a significant difference between the CO group and the BC group on the 21st day and the 28th day after exposure (P0.01). The time of exercise in the fourth quadrant was shortened in CO group after exposure, and there was significant difference between CO group and BC group and AC group in 14 days and 28 days (P0.05). The number of crossing the platform in CO group was lower than that in the other two groups after 28 days of exposure (P0.05). The number of pyramidal cells in hippocampus decreased after acute CO poisoning. Apoptosis of pyramidal cells in hippocampal area of rats in the 30% CO group was detected by Tunel method. Apoptosis began to increase on the 3rd day and reached the peak on the 7th day and reached the peak at the end of 28 days, and there was still a small amount of apoptosis in the hippocampal pyramidal cells of the rats in the control group. Compared with BC group and AC group, the apoptotic index at each time point had significant difference (P0.01) .4.The result of immunohistochemical staining was: 1) the expression of HIF-1 偽 protein in BC group and AC group was lower than that in BC group and AC group, and the expression of HIF-1 偽 protein was lower in BC group and AC group than in BC group and AC group. The expression of HIF-1 偽 in CO group was significantly higher than that in BC group and AC group (P0.01), and the expression of HIF-1 偽 in CO group was significantly higher than that in BC group and AC group (P0.01), and the expression of HIF-1 偽 in CO group was significantly higher than that in BC group and AC group (P0.01), and the expression of HIF-1 偽 in CO group was significantly higher than that in BC group (P0.01). 2) determination of EPO protein: there was a little expression of EPO protein in BC group and AC group, and there was no significant difference between different time points. The level of EPO protein in CO group was significantly higher than that in BC group and AC group at 1 day after exposure to CO for 1 day, and decreased at the peak of 3 days and 7 days after exposure to CO (P0.01), and there was a significant difference between CO group and AC group at 14 days after exposure (P0.01). Conclusion 1. The method of intraperitoneal injection of CO to make DEACMP rat model is simple, which is an ideal animal model for DEACMP. 2. Morris water maze test is a reliable basis for preliminary judgement of DEACMP in rats. 3. HIF-1 偽 may be involved in the pathogenesis of DEACMP by inducing delayed apoptosis of hippocampal neurons after acute CO poisoning. 4. The neuroprotective effect of EPO in the middle and late stages of acute CO poisoning may be one of the causes of DEACMP.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R747.9

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