老年期痴呆同病异证的Tau蛋白磷酸化研究

发布时间：2018-01-19 13:45

  本文关键词： 老年期痴呆 Tau蛋白磷酸化 同病异证 蛋白机制　出处：《山西中医学院》2016年硕士论文　论文类型：学位论文

【摘要】：目的:探寻研究老年期痴呆肾虚精亏证、痰浊阻窍证、寒凝血瘀证三个典型证候在临床常见的6个磷酸化位点(Thr205、Thr181、ser396、ser262、ser404、ser231)上Tau蛋白磷酸化的差异性。方法:本课题继承并改进前期造模方法,运用中医病因学理论结合现代医学相关理论知识构建了老年期痴呆的疾病模型;通过病证结合的方法,在动态过程中叠加相关的处理因素,分别施加房事不节和过度疲劳、投喂高脂饲料、寒冷刺激,在疾病模型基础上,建立老年期痴呆病证结合三个典型证候的动物模型,并通过行为学及血液生化检测对模型进行相关验证。随后,分别进行蛋白免疫印迹实验和免疫组化实验两个方法检测模型大鼠海马各蛋白位点的磷酸化水平。其中,对模型大鼠中的一部分进行蛋白免疫印迹的检测。提取海马组织蛋白,并定量;然后进行配胶、加样、跑胶、转膜、抗原抗体反应;通过一种影像分析的系统对条带的显影进行扫描纪录。实验得到的条带用GelPro 3.2软件进行灰度扫描和统计,对比各组目标条带之间的异同。对另一部分模型大鼠进行免疫组化实验。通过采用心脏灌注的方法对海马组织进行固定,制成切片经抗原抗体反应后,在光学显微镜下观察各位点细胞突触的阳性变化。通过IPP软件进行统计分析,比较各组间结果的异同。结果:1.老年期痴呆病证结合大鼠模型在行为学以及血液生化检测方面得到了相应的验证。在行为学的测试中与正常组的比较结果显示,其他组大鼠的逃避潜伏期均明显延长,其停留在目标象限的时间和穿越原平台区域的次数均有明显的减少,具有统计意义(P0.05),表明模型大鼠学习能力下降记忆能力减弱,与老年期痴呆疾病临床表现不符合。而在血液生化方面,肾虚精亏证组的睾酮含量明显高于其他各组,寒凝血瘀证组的血流变数值高于其他组,痰浊阻窍证组的血脂含量明显比其他组高,通过这些结果在一定程度上证明了我们构建的病证结合模型是成功的。2.老年期痴呆病证结合模型大鼠各蛋白位点磷酸化水平表达不一。在262位上各证候并未表现出明显的差异性;在181位上,肾虚精亏证组的磷酸化明显高于其他的组,痰浊阻窍证组的水平次之;在205位上,肾虚精亏证组和寒凝血瘀证组的水平升高的水平高于其他组;在231位上,表达最高的是寒凝血瘀证组,其次肾虚精亏证组较另外几组也有明显的提高;在396位上,寒凝血瘀证组的磷酸化水平最高,其他组磷酸化水平差异不大;在404位上,其他组磷酸化水平低于肾虚精亏证组和寒凝血瘀证组。3.在免疫组化阳性面积方面,在181位上,肾虚精亏证组的磷酸化阳性面积明显高于其他组,痰浊阻窍证组磷酸化阳性面积次之;在205位点上的肾虚精亏证组的阳性面积明最高;在231位上,阳性面积表达最高的是寒凝血瘀证组;在396位上,寒凝血瘀证组的磷酸化阳性面积表达水平最高,肾虚精亏证组次之;在404位上,肾虚精亏证组与寒凝血瘀证组的磷酸化水平明显高于其他组。4.在免疫组化的IOD值方面,在181位上,肾虚精亏证组的磷酸化IOD值高于其他组;在205位上,肾虚精亏证组阳性值也高于其他各组;在231位上,IOD值最高的是寒凝血瘀证组;在396位上,寒凝血瘀证组的磷酸化IOD值表达水平最高,肾虚精亏证组IOD值次之;在404位上,寒凝血瘀证组的IOD值明比其他组显高。结论:1.三个典型证候模型大鼠Tau的磷酸化均具有特异性,其中肾虚精亏证组各位点的磷酸化水平均有显著升高,而寒凝血瘀证组磷酸化水平主要集中在C末端的396,404位点上,痰浊阻窍证组主要在181和231位磷酸化水平有较明显的提高。提示我们在老年期痴呆不同的证候中,Tau蛋白与微管的结合能力降低,神经纤维缠结增多。2.老年期痴呆疾病的病理产物显示存在于脑中NFT的主要蛋白Tau的磷酸化,而由于不同的病因导致了Tau的磷酸化具有不同的表现,从基本的蛋白层面讨论中医学中同病异证的内涵的科学性。3.肾虚精亏证组的Tau蛋白位磷酸化发生位点最为广泛,且常常多发于脯氨酸富集区域,此区域的磷酸化会直接影响C末端的磷酸化,表明了肾虚精亏是老年期痴呆疾病的基本病机。而寒凝血瘀证组磷酸化发生的幅度最大,最为严重,且在C末端发生最为明显,表明了血瘀可能是老年期痴呆的最终病理产物。说明了三个证候因素互为病因,从而反映中医理论中因痰致瘀、因瘀致痰,因虚致实、实中有虚的特点。
[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，

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