發(fā)布時間：2018-10-20 18:29

【摘要】：背景 阿爾茨海默病(Alzheimer's disease, AD)是老齡人群中最常見也是最重要的一種退變性神經(jīng)疾病,臨床上以進行性的認知功能障礙,特別是空間學(xué)習(xí),記憶力障礙為主要特點。隨人類預(yù)期壽命的增加,此種毀滅性疾病發(fā)病率逐年上升,已繼心臟病,腫瘤,中風(fēng)后成為人類疾病死亡的第四大病因。大量研究證明阿爾茨海默病的特征性病理改變與腦前葉和海馬的老年斑沉積,神經(jīng)纖維纏結(jié)和神經(jīng)元凋亡緊密相連。但總體來說,阿爾茨海默病的發(fā)病機理仍不明確,且此種疾病目前尚無法徹底治愈。因此仍需要進行大量的研究。目前已有研究表明阿爾茨海默病的學(xué)習(xí),記憶能力的進行性退化與突觸功能障礙息息相關(guān),而突觸的結(jié)構(gòu)和功能也被認為在病理上與認知功能障礙關(guān)系最為緊密。因此,突觸功能障礙是改善認知功能障礙,延緩阿爾茨海默病發(fā)展的一個重要的治療靶點。鹽酸法舒地爾(Fasudil hydrochloride, FH)是法舒地爾的一種活性代謝產(chǎn)物。它是一個經(jīng)典的Rho激酶(Rho kinase, ROCK)的拮抗劑。已有報道法舒地爾能改善老齡大鼠,腦缺血模型大鼠的認知功能障礙。然而,鹽酸法舒地爾是否在阿爾茨海默病動物模型中同樣具有抗癡呆作用仍不明了,而其抗癡呆作用的具體機制更鮮有研究。在本研究中,我們運用側(cè)腦室注射鏈脲佐菌素(streptozotocin, STZ)的大鼠模型來觀測鹽酸法舒地爾對其學(xué)習(xí),記憶能力的作用,以及其具體機制。 目的 本研究討論了AD大鼠中空間學(xué)習(xí),記憶能力缺損與海馬突觸改變的聯(lián)系,以及通過檢測STZ組大鼠學(xué)習(xí),記憶能力,海馬CA1區(qū)顯微結(jié)構(gòu)變化,突觸囊泡素(synaptophysin, SYP)基因,蛋白表達和LIMK2, cofilin蛋白表達以及鹽酸法舒地爾對上述指標的作用探討鹽酸法舒地爾抗癡呆作用的具體分子機制。本文為日后鹽酸法舒地爾用于阿爾茨海默病的治療提供的寶貴的理論基礎(chǔ)。 方法 48只重250-300g的雄性SD大鼠由中南大學(xué)動物實驗中心提供,并被隨機分為4組(每組12只)：(1)假手術(shù)組(control);(2)鹽酸法舒地爾對照組(sham+FH);(3)模型組(STZ)；(4)鹽酸法舒地爾干預(yù)組(STZ+FH)。STZ組和STZ+FH組大鼠在第1天和第3天側(cè)腦室注射STZ兩次(1.5mg/kg體重),control組和sham+FH組大鼠給予等量的檸檬酸／檸檬酸鈉緩沖液。Sham+FH和STZ+FH組大鼠腹腔注射鹽酸法舒地爾(10mg/kg體重)4周,STZ組和control組大鼠則腹腔注射等量的生理鹽水。學(xué)習(xí)和記憶能力用Morris水迷宮檢測。HE染色和尼式染色檢測海馬CA1區(qū)神經(jīng)元的形態(tài)。顯微電鏡觀測海馬CA1區(qū)突觸顯微結(jié)構(gòu)。海馬CA1區(qū)Gray I型突觸界面參數(shù)用透射電鏡觀測及相關(guān)軟件測量分析。real-time PCR和western blotting分別檢測SYP基因和蛋白表達。LIMK2口cofilin的蛋白磷酸化水平也由western blotting檢測得出。運用SPSS18.0軟件對實驗數(shù)據(jù)進行統(tǒng)計學(xué)分析。用均值±標準差的方式表示計量結(jié)果。以重復(fù)測量的兩因素方差分析法統(tǒng)計分析Morris水迷宮中上臺潛伏期,游泳速度,當總體方差分析有差異時,LSD法行組間兩兩比較分析組間差異。穿越平臺次數(shù)用Wilcoxon signed-rank sum檢驗。其余變量用單因素方差分析,總體方差分析有差異時組間差異顯著性用LSD行組間兩兩比較。P值0.05為有統(tǒng)計學(xué)意義。 結(jié)果 1.與control組相比,STZ組大鼠在第2天到第5天的上臺潛伏期明顯延長,表明學(xué)習(xí)能力受損；靶向限游泳時間和穿越平臺次數(shù)明顯下降,表明記憶能力受損。鹽酸法舒地爾干預(yù)后,STZ+FH組大鼠中上述指標明顯改善。Sham+FH組大鼠在Morris水迷宮檢測中表現(xiàn)和control組大致相同,表明鹽酸法舒地爾本身對假手術(shù)大鼠并無學(xué)習(xí),記憶能力改善作用。 2.與control組相比,STZ組大鼠海馬CA1區(qū)神經(jīng)元數(shù)量減少且排列不規(guī)則,神經(jīng)元細胞器顯微結(jié)構(gòu)明顯受損。鹽酸法舒地爾干預(yù)后,STZ+FH組大鼠海馬CA1區(qū)神經(jīng)元數(shù)量明顯增加且排列較規(guī)則,神經(jīng)元細胞器的顯微結(jié)構(gòu)破壞程度也有所改善。 3.與control組相比,STZ組大鼠海馬SYP蛋白,基因表達明顯下降,而STZ+FH組大鼠SYP基因,蛋白表達有所回升。 4.與control組相比,STZ組大鼠突觸數(shù)量下降,伴有凹形突觸,穿孔型突觸比例下降。此外,還出現(xiàn)突觸間隙變窄,PSD厚度變薄,突觸活性帶長度縮短,突觸曲率降低改變。而鹽酸法舒地爾干預(yù)后上述突觸結(jié)構(gòu)退行性改變均有所改善。 5.與control組相比,STZ組大鼠LIMK2和cofilin磷酸化水平有所上調(diào)。而鹽酸法舒地爾能明顯下調(diào)STZ+FH組大鼠LIMK2和cofilin的磷酸化水平。 結(jié)論 1.大鼠側(cè)腦室注射STZ是一種阿爾茨海默病的動物模型。具有良好的可復(fù)制性和穩(wěn)定性,是日后阿爾茨海默病研究中一種比較理想的AD動物模型。 2.STZ所致的海馬神經(jīng)元和突觸結(jié)構(gòu)異常可能是導(dǎo)致模型組大鼠認知功能障礙的原因之一。鹽酸法舒地爾可能通過改善神經(jīng)元和突觸結(jié)構(gòu)和功能起到改善STZ+FH組大鼠學(xué)習(xí)和記憶能力的作用。 3.鹽酸法舒地爾所致的LIMK2和cofilin脫磷酸化過程可能是其保護突觸結(jié)構(gòu)和功能的分子機制之一。
[Abstract]:Background Alzheimer's disease (AD) is one of the most common and most important degenerative neurological diseases in the elderly population, which is mainly characterized by progressive cognitive dysfunction, especially spatial learning and memory disorder. Characteristic. With the increase of human life expectancy, the incidence of such destructive disease increases year by year, and has become the fourth major cause of human disease after heart disease, tumor and stroke. Etiology: A large number of studies have shown that the characteristic pathological changes of Alzheimer's disease are closely related to senile plaque deposits, nerve fiber entanglement and neuronal apoptosis in the anterior lobe and hippocampus of the brain. But in general, the pathogenesis of Alzheimer's disease is still unclear, and the disease is not yet complete It's cured. So there's still a lot to be done. At present, studies have shown that the study of Alzheimer's disease and the progressive degradation of memory ability are closely related to synaptic dysfunction, and the structure and function of synapses are also considered to be the most important in the pathogenesis of Alzheimer's disease Therefore, synaptic dysfunction is an important treatment for improving cognitive dysfunction and delaying the development of Alzheimer's disease. Target. Huudil hydrochloride (FH) is a kind of active metabolism in the method. Product. It is a classic Rho kinase (ROCK). Anti-agent. It has been reported that Shuisher can improve the cognitive function of aged rats and rats with cerebral ischemia model. However, it is unclear whether the anti-dementia effect is still unknown in the animal model of Alzheimer's disease, and the specific mechanism of anti-dementia effect is less. In this study, we used the rat model of streptozoocin (STZ) with lateral ventricular injection to observe the effect of hydrochloric acid method on its learning and memory ability, as well as its specific function. mechanism Objective: To study the relation between spatial learning and memory ability defect in AD rats and synaptic changes in hippocampus, and to study the changes in microstructure of hippocampal CA1 region, synaptophin (SYP) gene and protein by detecting the learning and memory ability of STZ rats. Expression of the expression and expression of LIMK2, cofilin protein and the effect of hydrochloric acid method on the above-mentioned indexes to explore the effect of hydrochloric acid method on anti-dementia Specific molecular mechanisms for use in the treatment of Alzheimer's disease. Bao's treasure Methods 48 male Sprague-Dawley rats weighing 250-300g were randomly divided into 4 groups (12 rats in each group): (1) sham operation group (control); (2) sham operation group (sham + F). H); (3) model group (STZ); (4) treatment group (STZ + FH). STZ group and STZ + FH group rats were injected STZ twice (1. 5mg/ kg body weight) on day 1 and day 3, control group and sham + FH group rats were given. An equal amount of citric acid/ sodium citrate buffer. Shams + FH and STZ + FH rats were injected into the abdominal cavity of rats for 4 weeks, STZ group and control group. Rats were injected intraperitoneally with equal amounts of physiological saline. Learning and memory Ability to detect with Morris water maze. HE staining and nintedanib Morphological and microscopic electron microscopic examination of neurons in hippocampal CA1 region Observation of synaptic microstructure in CA1 region of hippocampus of hippocampal CA1 region in hippocampal CA1 region Radio Mirror Observation and Related Software Measurement Analysis The expression of SYP gene and protein was detected. rn blotting detection results. SPSS18. 0 Statistical analysis of experimental data was performed by software. The method of variance analysis of two factors was used to analyze the incubation period and swimming speed in Morris water maze. Difference between the two comparative analysis groups between the two groups. Wilcoxon si was used for the number of crossing platforms. Received-Cadsum test. The remaining variables were analyzed by single-factor analysis of variance, and there was a difference in the overall variance of variance. Comparison between the two groups in the LSD line group .P Results 1. Compared with control group, the incubation period of STZ group in STZ group was significantly prolonged on day 2 to day 5, indicating impaired learning ability and limited swimming time. and the number of crossing platforms is obviously reduced, the memory capacity is impaired, After that, the above indexes in STZ + FH group were obviously improved. In the Morris water maze test, the rats were almost the same in the sham + FH group. The number of neurons in hippocampal CA1 region of STZ group was higher than that of control group. The number of neurons in hippocampal CA1 region of STZ + FH group was obviously increased and the arrangement was higher than that of group STZ + FH group. Compared with control group, the expression of SYP protein and gene in hippocampus of STZ group was obviously lower than that of control group. Compared with control group, the SYP gene and protein expression of STZ + FH group increased. The number of synapses in the STZ group decreased, accompanied by a decrease in the synapse of the concave synapse and the perforation type. In addition, synaptic cleft also appeared. Narnarrowed, reduced PSD thickness, reduced synaptic activity band length, reduced synaptic curvature The changes of the postsynaptic structure were improved after the intervention of the hydrochloric acid method, and the changes of the postsynaptic structure were improved. Compared with the group, the phosphorylation level of LIMK2 and cofilin in STZ group was up-regulated. Obvious Down-regulation of LIMK2 and cofilin in STZ + FH Rats Conclusion 1. Intracerebroventricular injection of STZ in rats is an animal model of Alzheimer's disease. There is good replicability and stability, which is an ideal AD animal model in the study of Alzheimer's disease. 2. The abnormality of hippocampal neurons and synaptic structures caused by STZ may be one of the causes of cognitive dysfunction in the model group. It is possible to improve the learning and memory ability of STZ + FH rats by improving the structure and function of neurons and synapses.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R749.16

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本文編號：2284021