HIF-1α及EPO在急性一氧化碳中毒迟发性脑病大鼠海马区表达的实验研究

发布时间：2018-07-17 00:00

【摘要】：目的：通过建立急性CO中毒迟发型脑病（DEACMP）大鼠模型，观察HIF-1α及EPO在相应动物模型中的表达变化，探讨HIF-1α及EPO在DEACMP发病机制中的作用。方法：78只健康雄性成年SD大鼠（SPF级），体重在240～280g之间，随机分为三组：空白对照组（BC组）18只、空气对照组（AC组）30只、急性一氧化碳中毒迟发性脑病组（CO组）30只。其中CO组采用腹腔注射法建立DEACMP大鼠模型，AC组采用腹腔注射法注射等量空气，BC组不进行任何手术操作。按中毒后1d、3d、7d、14d、21d、28d六个时间点将三个组各分为6个亚组，其中CO组和AC组每个亚组5只大鼠，BC组每个亚组3只大鼠。观察大鼠急性CO中毒表现和大鼠动脉血COHb浓度及中毒后期运用Morris水迷宫实验检测大鼠认知行为学改变。各组大鼠的脑组织切片均应用HE染色观察大鼠海马区病理变化，用TUNEL染色检测海马区锥体细胞凋亡情况，应用免疫组化染色检测海马区HIF-1α、EPO蛋白的表达。结果：1、Morris水迷宫实验结果：CO组大鼠在染毒后出现平均逃避潜伏期延长，且在染毒后14d、21d及28d时，CO组较BC组和AC组比较差异均有显著统计学意义（P0.01)。染毒后CO组大鼠在第Ⅳ象限运动时间有缩短趋势，且染毒14d及染毒28d时，CO组较BC组及AC组比较差异有统计学意义（P0.05)。染毒后CO组大鼠穿越平台次数有降低趋势，染毒28d时，CO组穿越平台次数较其余两组低，差异有统计学意义（P0.05）。2、HE染色：CO组大鼠在急性CO中毒后出现海马区锥体细胞数目减少、细胞排列松散、细胞水肿、核固缩、核碎裂等病理学改变。3、TUNEL法检测：CO组大鼠海马区锥体细胞于染毒3d时凋亡开始增多，，7d时达到高峰，28d时仍有少量细胞凋亡，各时间点凋亡指数与BC组及AC组比较，差异具有显著性（P0.01)。4、免疫组化染色结果：1）HIF-1α蛋白测定：HIF-1α在BC组及AC组中表达较少，且各时间点间差异不具有显著性，CO组在染毒1d时出现HIF-1α的表达升高，3d达到峰值，28d时仍有表达，表达量明显高于BC组及AC组，差异具有显著性（P0.01）；2）EPO蛋白测定：在BC组及AC组中有少量表达，且各时间点间差异不具有显著性，CO组在染毒1d时明显升高，3d为高峰，7d时下降，染毒1d、3d、7d、14d时CO组较BC组与AC组差异具有显著性（P0.01）。结论：1.腹腔注射CO法制作DEACMP大鼠模型，操作简单，是研究DEACMP较为理想的动物模型；2.Morris水迷宫实验是初步判断大鼠DEACMP较为可靠的依据；3.HIF-1α可能通过诱导急性CO中毒后海马区神经元的迟发性凋亡而参与DEACMP的发病；4.EPO在急性CO中毒后中后期神经保护作用减弱，可能是导致DEACMP发病原因之一。
[Abstract]:Objective: to investigate the role of HIF-1 伪 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 伪 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 伪 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 伪 protein in BC group and AC group was lower than that in BC group and AC group, and the expression of HIF-1 伪 protein was lower in BC group and AC group than in BC group and AC group. The expression of HIF-1 伪 in CO group was significantly higher than that in BC group and AC group (P0.01), and the expression of HIF-1 伪 in CO group was significantly higher than that in BC group and AC group (P0.01), and the expression of HIF-1 伪 in CO group was significantly higher than that in BC group and AC group (P0.01), and the expression of HIF-1 伪 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 伪 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.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R747.9

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