本文關(guān)鍵詞： 老年期癡呆 Tau蛋白磷酸化 同病異證 蛋白機制　出處：《山西中醫(yī)學(xué)院》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探尋研究老年期癡呆腎虛精虧證、痰濁阻竅證、寒凝血瘀證三個典型證候在臨床常見的6個磷酸化位點(Thr205、Thr181、ser396、ser262、ser404、ser231)上Tau蛋白磷酸化的差異性。方法:本課題繼承并改進前期造模方法,運用中醫(yī)病因?qū)W理論結(jié)合現(xiàn)代醫(yī)學(xué)相關(guān)理論知識構(gòu)建了老年期癡呆的疾病模型;通過病證結(jié)合的方法,在動態(tài)過程中疊加相關(guān)的處理因素,分別施加房事不節(jié)和過度疲勞、投喂高脂飼料、寒冷刺激,在疾病模型基礎(chǔ)上,建立老年期癡呆病證結(jié)合三個典型證候的動物模型,并通過行為學(xué)及血液生化檢測對模型進行相關(guān)驗證。隨后,分別進行蛋白免疫印跡實驗和免疫組化實驗兩個方法檢測模型大鼠海馬各蛋白位點的磷酸化水平。其中,對模型大鼠中的一部分進行蛋白免疫印跡的檢測。提取海馬組織蛋白,并定量;然后進行配膠、加樣、跑膠、轉(zhuǎn)膜、抗原抗體反應(yīng);通過一種影像分析的系統(tǒng)對條帶的顯影進行掃描紀錄。實驗得到的條帶用GelPro 3.2軟件進行灰度掃描和統(tǒng)計,對比各組目標條帶之間的異同。對另一部分模型大鼠進行免疫組化實驗。通過采用心臟灌注的方法對海馬組織進行固定,制成切片經(jīng)抗原抗體反應(yīng)后,在光學(xué)顯微鏡下觀察各位點細胞突觸的陽性變化。通過IPP軟件進行統(tǒng)計分析,比較各組間結(jié)果的異同。結(jié)果:1.老年期癡呆病證結(jié)合大鼠模型在行為學(xué)以及血液生化檢測方面得到了相應(yīng)的驗證。在行為學(xué)的測試中與正常組的比較結(jié)果顯示,其他組大鼠的逃避潛伏期均明顯延長,其停留在目標象限的時間和穿越原平臺區(qū)域的次數(shù)均有明顯的減少,具有統(tǒng)計意義(P0.05),表明模型大鼠學(xué)習(xí)能力下降記憶能力減弱,與老年期癡呆疾病臨床表現(xiàn)不符合。而在血液生化方面,腎虛精虧證組的睪酮含量明顯高于其他各組,寒凝血瘀證組的血流變數(shù)值高于其他組,痰濁阻竅證組的血脂含量明顯比其他組高,通過這些結(jié)果在一定程度上證明了我們構(gòu)建的病證結(jié)合模型是成功的。2.老年期癡呆病證結(jié)合模型大鼠各蛋白位點磷酸化水平表達不一。在262位上各證候并未表現(xiàn)出明顯的差異性;在181位上,腎虛精虧證組的磷酸化明顯高于其他的組,痰濁阻竅證組的水平次之;在205位上,腎虛精虧證組和寒凝血瘀證組的水平升高的水平高于其他組;在231位上,表達最高的是寒凝血瘀證組,其次腎虛精虧證組較另外幾組也有明顯的提高;在396位上,寒凝血瘀證組的磷酸化水平最高,其他組磷酸化水平差異不大;在404位上,其他組磷酸化水平低于腎虛精虧證組和寒凝血瘀證組。3.在免疫組化陽性面積方面,在181位上,腎虛精虧證組的磷酸化陽性面積明顯高于其他組,痰濁阻竅證組磷酸化陽性面積次之;在205位點上的腎虛精虧證組的陽性面積明最高;在231位上,陽性面積表達最高的是寒凝血瘀證組;在396位上,寒凝血瘀證組的磷酸化陽性面積表達水平最高,腎虛精虧證組次之;在404位上,腎虛精虧證組與寒凝血瘀證組的磷酸化水平明顯高于其他組。4.在免疫組化的IOD值方面,在181位上,腎虛精虧證組的磷酸化IOD值高于其他組;在205位上,腎虛精虧證組陽性值也高于其他各組;在231位上,IOD值最高的是寒凝血瘀證組;在396位上,寒凝血瘀證組的磷酸化IOD值表達水平最高,腎虛精虧證組IOD值次之;在404位上,寒凝血瘀證組的IOD值明比其他組顯高。結(jié)論:1.三個典型證候模型大鼠Tau的磷酸化均具有特異性,其中腎虛精虧證組各位點的磷酸化水平均有顯著升高,而寒凝血瘀證組磷酸化水平主要集中在C末端的396,404位點上,痰濁阻竅證組主要在181和231位磷酸化水平有較明顯的提高。提示我們在老年期癡呆不同的證候中,Tau蛋白與微管的結(jié)合能力降低,神經(jīng)纖維纏結(jié)增多。2.老年期癡呆疾病的病理產(chǎn)物顯示存在于腦中NFT的主要蛋白Tau的磷酸化,而由于不同的病因?qū)е铝薚au的磷酸化具有不同的表現(xiàn),從基本的蛋白層面討論中醫(yī)學(xué)中同病異證的內(nèi)涵的科學(xué)性。3.腎虛精虧證組的Tau蛋白位磷酸化發(fā)生位點最為廣泛,且常常多發(fā)于脯氨酸富集區(qū)域,此區(qū)域的磷酸化會直接影響C末端的磷酸化,表明了腎虛精虧是老年期癡呆疾病的基本病機。而寒凝血瘀證組磷酸化發(fā)生的幅度最大,最為嚴重,且在C末端發(fā)生最為明顯,表明了血瘀可能是老年期癡呆的最終病理產(chǎn)物。說明了三個證候因素互為病因,從而反映中醫(yī)理論中因痰致瘀、因瘀致痰,因虛致實、實中有虛的特點。
[Abstract]:Objective: To explore the study of senile dementia in kidney deficiency syndrome, phlegm syndrome, cold coagulation and blood stasis type three typical syndromes in 6 clinical common phosphorylation sites (Thr205, Thr181, ser396, ser262, ser404, ser231) the difference on the phosphorylation of Tau. Methods: the subject of inheritance early and improved modeling method, using the theory of TCM etiology construct disease model of senile dementia combined with modern medical theory of knowledge; method through the combination of disease and syndrome, in the dynamic process of superposition related factors, are applied in sexual life and fatigue, fed with high fat diet, cold stimulation, in the disease model on the basis of an animal model of three typical syndromes of a dementia syndrome, and through the behavior and blood biochemical detection of the model were verified. Then, respectively by Western blot and immunohistochemical experiments with two methods The phosphorylation level was detected in the rat model of each protein loci were detected. The Western blot on the part of the model in rats. Hippocampus protein extraction, and quantitative; then with glue, and glue, run, transfer film, antigen antibody reaction; through a shadow image analysis system the bands were scanned. The record of developing bands with GelPro 3.2 software for gray scale scanning and statistics, comparison between groups of target band. In another part of the rat model by immunohistochemical experiment. By using the method of heart perfusion on hippocampal tissues were fixed, sectioned by antigen antibody reaction after observing the positive changes of each cell synapses under optical microscope. Statistical analysis was performed by IPP software, and the results of each group were compared. Results: 1. senile dementia disease combined with syndrome rat models and in blood In terms of liquid biochemical detection have been proposed and verified. The comparison shows that with the normal group in behavioral tests, other rats escape latency were significantly prolonged, the residence time in the target quadrant and the frequency of crossing the original platform was significantly reduced, with statistical significance (P0.05), showed that the model in the learning ability and memory ability weakened, is not consistent with clinical manifestations of dementia in old age. Blood biochemistry, testosterone deficient kidney essence syndrome group was significantly higher than other groups, the blood flow value of cold coagulation and blood stasis type were higher than other groups, blood lipid content of phlegm syndrome group was significantly better than the other group high, through these results in a certain extent, it is proved that the model combining disease with syndrome we constructed.2. dementia syndrome successfully with the protein phosphorylation in a rat model of expression is not a 262 on each. 璇佸，

本文編號：1444453