G-CSF对Alzheimer病模型大鼠的治疗及其相关凋亡因子的影响

发布时间：2018-06-13 03:30

本文选题：阿尔茨海默病 + 粒细胞集落刺激因子　；参考：《吉林大学》2012年硕士论文

【摘要】：阿尔茨海默病(Alzheimer disease，AD)是一种老年人常见的神经系统变性疾病。AD的典型组织病理学特征为老年斑、神经元纤维缠结及神经元缺失。其起病隐袭，发病早期不易被发现，临床上常呈持续进展的智能减退，多伴人格障碍。在发达国家，AD是位于心脏病、肿瘤及中风之后的第四位致死性疾病，随年龄的逐渐增长，患者量的不断增加势必会给社会及家庭带来更大的负担。如何有效地治疗AD已成为社会广泛关注的问题。目前AD的病因及发病机制尚未阐明，亦无特效的治疗办法。细胞凋亡与AD的发生、发展密切相关。粒细胞集落刺激因子（G-CSF）具有促进增殖分化、抗凋亡、抗炎等作用。近年来发现在脑缺血动物模型的治疗实验中，G-CSF具有神经保护作用。但目前用于治疗AD的研究较少。本实验旨在观察G-CSF对AD模型大鼠的治疗作用及其对相关凋亡因子的影响。为AD的治疗研究提供有益的帮助。本实验大体步骤如下，（1）首先用Morris水迷宫筛选反应迅速、活跃的Wistar雄性大鼠30只，体重250～300g，鼠龄3-4个月，分笼喂养。（2）随机分为三组：正常组，模型对照组，G-CSF治疗组。每组10只。（3）参照大鼠脑立体定位图谱，应用大鼠脑立体定向仪切除双侧穹窿-海马伞制备AD大鼠模型。（4）术后第14天开始连续5天行Morris水迷宫定位航行训练，记录逃避潜伏期成绩，取5天内的平均数，以期模型对照组及G-CSF治疗组明显高于正常组，证明建模成功。（5）建模成功后，正常组及模型对照组向每只大鼠腹腔注射0.3ml/kg·d磷酸盐缓冲液（PBS），G-CSF治疗组给予每只大鼠腹腔注射50ug（0.3ml）/kg·d G-CSF，均连续注射7天。从给药第1天起，用药后第10天开始再次行Morris水迷宫训练，前5天进行定位航行实验，记录各组5天内逃避潜伏期成绩，取平均数。最后一天撤除平台，行空间探索实验，记录各组目的象限游泳距离百分比。（6）测试完成后处死三组大鼠，灌注取脑，制备石蜡切片，行HE染色观察各组大鼠脑组织病理改变，应用免疫组化方法检测各组脑皮质及海马区凋亡因子Bcl-2、Caspase-3的变化。（7）采集图像，免疫阳性细胞数计数，，整理数据，所有数据采用SPSS11.0统计学软件进行处理，以均数±标准差（x±s）表示，采用方差分析及两两比较t检验进行数据分析，P0.05表示差异具有统计学意义。结果显示，（1）术后14天进行的连续5天Morris水迷宫实验结果表明，模型对照组及G-CSF治疗组与正常组相比，其逃避潜伏期均明显延长(P0.01)；模型对照组与G-CSF治疗组比较逃避潜伏期，差异不具有统计学意义(P0.05)，证明建模成功。（2）给予G-CSF治疗后第10天开始再次连续5天行Morris水迷宫训练，正常组与模型对照组相比，逃避潜伏期明显缩短（P0.01），正常组与G-CSF治疗组之间相比，逃避潜伏期明显缩短（P0.05），G-CSF治疗组与模型对照组相比，逃避潜伏期明显缩短（P0.05）。最后一天行空间探索实验，记录目的象限游泳距离百分比（目的象限百分比），正常组与模型对照组相比，目的象限百分比明显延长（P0.01），G-CSF治疗组与模型对照组相比，目的象限百分比明显延长（P0.05），正常组与G-CSF治疗组相比，目的象限百分比明显延长（P0.05）。（3）大鼠脑组织各区病理学超微结构观察：常规HE染色，正常组皮层、海马等结构完整，形态正常，细胞排列整齐，分布均匀。G-CSF治疗组皮层、海马等处可见少量胶质细胞增生，细胞凋亡改变少见。模型对照组皮层神经细胞损坏明显，可见大量散在的单个固缩凋亡细胞；海马锥体细胞带变稀、紊乱、中断，同时可见多个细胞体积缩小，胞质浓染，胞核固缩。（4）用免疫组化方法测定大鼠皮层及海马CA1区凋亡因子Bcl-2及Caspase-3的表达，分别计数阳性反应细胞数，阳性反应为胞浆呈黄色到棕褐色，有时也可见于胞膜和胞核。模型对照组与正常组相比，皮层及海马CA1区Bcl-2表达明显减少（P0.05），Caspase-3的表达明显增加（P0.05）；G-CSF治疗组与模型对照组相比，皮层及海马CA1区Bcl-2表达明显增加（P0.05），Caspase-3的表达明显减少（P0.05）。综上，本研究说明，G-CSF对切除双侧穹窿-海马伞制备的AD模型大鼠具有治疗作用，可以在一定程度上改善模型大鼠的认知障碍，而G-CSF通过上调皮层及海马区Bcl-2表达、下调Caspase-3表达的抗凋亡机制参与了对AD模型大鼠的这种治疗作用。
[Abstract]:Alzheimer disease (AD) is a common type of neurodegenerative disease of the elderly, the typical histopathological features of.AD, the senile plaques, neurofibrillary tangles and neuronal loss. AD is the fourth fatal disease after heart disease, tumor and stroke. With the increase of age, the increasing amount of patients will bring greater burden to society and family. How to effectively treat AD has become a widespread concern. At present, the etiology and pathogenesis of AD have not yet been clarified, and there is no special treatment. Method. Apoptosis is closely related to the development of AD. Granulocyte colony stimulating factor (G-CSF) has the role of promoting proliferation, differentiation, anti apoptosis and anti-inflammatory. In recent years, it has been found that G-CSF has neuroprotective effect in the treatment experiments of cerebral ischemia animal models. However, there are few studies on the treatment of AD at present. The aim of this experiment is to observe the AD model of G-CSF. The therapeutic effect of the rat and its effect on the related apoptosis factors will provide useful help for the research and treatment of AD.
The main steps of this experiment were as follows: (1) first, 30 male rats with rapid and active reaction were screened by Morris water maze, with a weight of 250 to 300g and 3-4 months of age for 3-4 months. (2) they were randomly divided into three groups: normal group, model control group, G-CSF treatment group, 10 rats in each group. (3) the rat brain stereotaxic atlas was applied to the stereotaxis of rat brain. AD rat model was prepared by bilateral fornix fornix parachute. (4) Morris water maze navigation training was performed on the fourteenth day after 5 days, and the average number of escape latency was recorded in 5 days. The model control group and the G-CSF treatment group were obviously higher than the normal group. (5) the normal group and the model control group after the successful modeling were successful. Each rat was intraperitoneally injected with 0.3ml/kg D phosphate buffer solution (PBS). The rats in the G-CSF treatment group were given 50ug (0.3ml) /kg. D G-CSF in each rat for 7 days. From the first day of the administration, the training of the Morris water maze was repeated on the tenth day after the drug administration, and the fixed navigation experiment was carried out on the first 5 days, and the escape latency was recorded in each group for 5 days, and the results of the escape latency were recorded in each group. The average number. In the last day, the platform was removed and the space exploration experiment was carried out to record the percentage of swimming distance in each group. (6) after the test was completed, three groups of rats were killed, the brain was perfused and the paraffin section was prepared. HE staining was used to observe the pathological changes in the brain tissue of each group, and the apoptosis factor Bcl-2 in the cortex and hippocampus of each group was detected by immunohistochemical method, C The change of aspase-3. (7) collect the image, count the number of immunoreactive cells, arrange the data, all the data are processed by SPSS11.0 statistics software, the mean number of standard deviation (x + s), analysis of variance and 22 comparison t test for data analysis, P0.05 indicates that difference has statistical significance.
The results showed that (1) the results of the 5 day Morris water maze test on the 14 day after the operation showed that the escape latency of the model control group and the G-CSF treatment group was significantly longer than that of the normal group (P0.01), and the model control group was compared with the G-CSF treatment group, and the difference was not statistically significant (P0.05), which proved that the modeling was successful. (2) G-CSF After tenth days after treatment, the Morris water maze was trained again for 5 days. The escape latency was significantly shortened (P0.01) in the normal group compared with the model control group. The escape latency was significantly shortened (P0.05) in the normal group compared with the G-CSF treatment group. The escape latency was significantly shortened (P0.05) in the G-CSF treatment group compared with the model control group. The last day was empty. Between the normal group and the model control group, the percentage of the target quadrant was significantly prolonged (P0.01). Compared with the model control group, the percentage of the target quadrant was significantly longer (P0.05) than the model control group. The percentage of the target quadrant was significantly prolonged in the normal group compared with the G-CSF group. (P0.05). (3) observation of pathological ultrastructure in all regions of brain tissue of rats: routine HE staining, normal group cortex, hippocampus and other structures were complete, normal morphology, and orderly cells, distributed evenly.G-CSF treatment group cortex, hippocampus and other areas can be seen in a small number of glial cells proliferation, cell apoptosis is rare. Model control group cortical neurons damage obvious, A large number of isolated apoptotic cells were found. The hippocampal pyramidal cells were dilute, disorderly, and disrupted. At the same time, multiple cells were reduced, cytoplasm concentrated and nucleus retracted. (4) the expression of apoptosis factor Bcl-2 and Caspase-3 in the cortex and hippocampal CA1 region of rats was measured by immunohistochemistry. The positive reaction cells were counted and the positive reaction was positive. In the model control group, the expression of Bcl-2 in the cortex and hippocampal CA1 area decreased significantly (P0.05) and the expression of Caspase-3 increased significantly (P0.05). Compared with the model control group, the expression of Bcl-2 in the cortex and the hippocampal CA1 area was significantly increased (P0.05), the expression of Caspase-3 in the G-CSF group. Significantly decreased (P0.05).
To sum up, this study shows that G-CSF has a therapeutic effect on AD model rats excised by bilateral fornix fornix parachute. It can improve cognitive impairment in model rats to a certain extent, while G-CSF is expressed in the upper crust and hippocampus Bcl-2, and the anti apoptotic mechanism of Caspase-3 expression is involved in this treatment of AD model rats.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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