本文選題：阿爾茨海默病 + 粒細(xì)胞集落刺激因子��； 參考：《吉林大學(xué)》2012年碩士論文

【摘要】：阿爾茨海默病(Alzheimer disease，AD)是一種老年人常見(jiàn)的神經(jīng)系統(tǒng)變性疾病。AD的典型組織病理學(xué)特征為老年斑、神經(jīng)元纖維纏結(jié)及神經(jīng)元缺失。其起病隱襲，發(fā)病早期不易被發(fā)現(xiàn)，臨床上常呈持續(xù)進(jìn)展的智能減退，多伴人格障礙。在發(fā)達(dá)國(guó)家，AD是位于心臟病、腫瘤及中風(fēng)之后的第四位致死性疾病，隨年齡的逐漸增長(zhǎng)，患者量的不斷增加勢(shì)必會(huì)給社會(huì)及家庭帶來(lái)更大的負(fù)擔(dān)。如何有效地治療AD已成為社會(huì)廣泛關(guān)注的問(wèn)題。目前AD的病因及發(fā)病機(jī)制尚未闡明，亦無(wú)特效的治療辦法。細(xì)胞凋亡與AD的發(fā)生、發(fā)展密切相關(guān)。粒細(xì)胞集落刺激因子（G-CSF）具有促進(jìn)增殖分化、抗凋亡、抗炎等作用。近年來(lái)發(fā)現(xiàn)在腦缺血?jiǎng)游锬Ｐ偷闹委煂?shí)驗(yàn)中，G-CSF具有神經(jīng)保護(hù)作用。但目前用于治療AD的研究較少。本實(shí)驗(yàn)旨在觀察G-CSF對(duì)AD模型大鼠的治療作用及其對(duì)相關(guān)凋亡因子的影響。為AD的治療研究提供有益的幫助。 本實(shí)驗(yàn)大體步驟如下，（1）首先用Morris水迷宮篩選反應(yīng)迅速、活躍的Wistar雄性大鼠30只，體重250～300g，鼠齡3-4個(gè)月，分籠喂養(yǎng)。（2）隨機(jī)分為三組：正常組，模型對(duì)照組，G-CSF治療組。每組10只。（3）參照大鼠腦立體定位圖譜，應(yīng)用大鼠腦立體定向儀切除雙側(cè)穹窿-海馬傘制備AD大鼠模型。（4）術(shù)后第14天開(kāi)始連續(xù)5天行Morris水迷宮定位航行訓(xùn)練，記錄逃避潛伏期成績(jī)，取5天內(nèi)的平均數(shù)，以期模型對(duì)照組及G-CSF治療組明顯高于正常組，證明建模成功。（5）建模成功后，正常組及模型對(duì)照組向每只大鼠腹腔注射0.3ml/kg·d磷酸鹽緩沖液（PBS），G-CSF治療組給予每只大鼠腹腔注射50ug（0.3ml）/kg·d G-CSF，均連續(xù)注射7天。從給藥第1天起，用藥后第10天開(kāi)始再次行Morris水迷宮訓(xùn)練，前5天進(jìn)行定位航行實(shí)驗(yàn)，記錄各組5天內(nèi)逃避潛伏期成績(jī)，取平均數(shù)。最后一天撤除平臺(tái)，行空間探索實(shí)驗(yàn)，記錄各組目的象限游泳距離百分比。（6）測(cè)試完成后處死三組大鼠，灌注取腦，制備石蠟切片，行HE染色觀察各組大鼠腦組織病理改變，應(yīng)用免疫組化方法檢測(cè)各組腦皮質(zhì)及海馬區(qū)凋亡因子Bcl-2、Caspase-3的變化。（7）采集圖像，免疫陽(yáng)性細(xì)胞數(shù)計(jì)數(shù)，，整理數(shù)據(jù)，所有數(shù)據(jù)采用SPSS11.0統(tǒng)計(jì)學(xué)軟件進(jìn)行處理，以均數(shù)±標(biāo)準(zhǔn)差（x±s）表示，采用方差分析及兩兩比較t檢驗(yàn)進(jìn)行數(shù)據(jù)分析，P0.05表示差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)果顯示，（1）術(shù)后14天進(jìn)行的連續(xù)5天Morris水迷宮實(shí)驗(yàn)結(jié)果表明，模型對(duì)照組及G-CSF治療組與正常組相比，其逃避潛伏期均明顯延長(zhǎng)(P0.01)；模型對(duì)照組與G-CSF治療組比較逃避潛伏期，差異不具有統(tǒng)計(jì)學(xué)意義(P0.05)，證明建模成功。（2）給予G-CSF治療后第10天開(kāi)始再次連續(xù)5天行Morris水迷宮訓(xùn)練，正常組與模型對(duì)照組相比，逃避潛伏期明顯縮短（P0.01），正常組與G-CSF治療組之間相比，逃避潛伏期明顯縮短（P0.05），G-CSF治療組與模型對(duì)照組相比，逃避潛伏期明顯縮短（P0.05）。最后一天行空間探索實(shí)驗(yàn)，記錄目的象限游泳距離百分比（目的象限百分比），正常組與模型對(duì)照組相比，目的象限百分比明顯延長(zhǎng)（P0.01），G-CSF治療組與模型對(duì)照組相比，目的象限百分比明顯延長(zhǎng)（P0.05），正常組與G-CSF治療組相比，目的象限百分比明顯延長(zhǎng)（P0.05）。（3）大鼠腦組織各區(qū)病理學(xué)超微結(jié)構(gòu)觀察：常規(guī)HE染色，正常組皮層、海馬等結(jié)構(gòu)完整，形態(tài)正常，細(xì)胞排列整齊，分布均勻。G-CSF治療組皮層、海馬等處可見(jiàn)少量膠質(zhì)細(xì)胞增生，細(xì)胞凋亡改變少見(jiàn)。模型對(duì)照組皮層神經(jīng)細(xì)胞損壞明顯，可見(jiàn)大量散在的單個(gè)固縮凋亡細(xì)胞；海馬錐體細(xì)胞帶變稀、紊亂、中斷，同時(shí)可見(jiàn)多個(gè)細(xì)胞體積縮小，胞質(zhì)濃染，胞核固縮。（4）用免疫組化方法測(cè)定大鼠皮層及海馬CA1區(qū)凋亡因子Bcl-2及Caspase-3的表達(dá)，分別計(jì)數(shù)陽(yáng)性反應(yīng)細(xì)胞數(shù)，陽(yáng)性反應(yīng)為胞漿呈黃色到棕褐色，有時(shí)也可見(jiàn)于胞膜和胞核。模型對(duì)照組與正常組相比，皮層及海馬CA1區(qū)Bcl-2表達(dá)明顯減少（P0.05），Caspase-3的表達(dá)明顯增加（P0.05）；G-CSF治療組與模型對(duì)照組相比，皮層及海馬CA1區(qū)Bcl-2表達(dá)明顯增加（P0.05），Caspase-3的表達(dá)明顯減少（P0.05）。 綜上，本研究說(shuō)明，G-CSF對(duì)切除雙側(cè)穹窿-海馬傘制備的AD模型大鼠具有治療作用，可以在一定程度上改善模型大鼠的認(rèn)知障礙，而G-CSF通過(guò)上調(diào)皮層及海馬區(qū)Bcl-2表達(dá)、下調(diào)Caspase-3表達(dá)的抗凋亡機(jī)制參與了對(duì)AD模型大鼠的這種治療作用。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.16

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5 王俊濤;G-CSF及褪黑素對(duì)膠質(zhì)瘤細(xì)胞增殖、遷移和侵襲的影響及機(jī)制研究[D];山東大學(xué);2012年

6 范佳;粒細(xì)胞集落刺激因子在急性腦缺血中的神經(jīng)保護(hù)作用的研究[D];吉林大學(xué);2005年

7 李德冠;P38 MAPK抑制劑聯(lián)合G-CSF對(duì)全身γ射線照射小鼠輻射損傷的實(shí)驗(yàn)治療研究[D];北京協(xié)和醫(yī)學(xué)院;2012年

8 郭曉玲;G-CSF誘導(dǎo)T淋巴細(xì)胞向TH2分化的機(jī)制研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2008年

9 姚志峰;G-CSF對(duì)壓力超負(fù)荷下小鼠心室重構(gòu)和心力衰竭的影響[D];復(fù)旦大學(xué);2008年

10 曹秋云;Alzheimer病腦PET、神經(jīng)心理測(cè)定及BPSD研究[D];復(fù)旦大學(xué);2003年

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