分拣蛋白相关受体L1（SORL1）基因异常与阿尔茨海默病的相关性研究

发布时间：2018-05-08 01:32

本文选题：阿尔茨海默病 + SORL1基因　；参考：《中国人民解放军医学院》2013年硕士论文

【摘要】：研究背景阿尔茨海默病（Alzheimer’s disease,AD）是一种起病隐袭的进行性发展的痴呆，是最常见的痴呆类型，约占老年期痴呆的2/3。AD的发病机制与遗传因素有关，主要涉及到细胞凋亡、细胞分化、细胞周期等。目前普遍认为，发病关键环节之一是β淀粉样蛋白在脑部神经元的异常积聚，形成“老年斑块”和“神经元纤维缠结”。近年研究与大脑相关的基因很多,认为利用分子生物学技术，分析基因结构变异和表达状态，从而对疾病在表型改变以前进行早期诊断。分拣蛋白相关受体L1基因（sortilin-related receptor1gene，SORL1）是与阿尔茨海默病的患病风险有关联的新发基因之一，在神经细胞内表达，作为分选蛋白受体穿梭于胞膜、胞质和高尔基体，参与神经细胞的物质转运和交换过程。目前欧美等国家通过该基因序列中的单核甘酸多态性进行对照研究，但SORL1基因在外周血中的表达研究较少，尤其是其突变及异常甲基化与晚发型阿尔茨海默病的相关性研究甚少。本课题组的前期研究证明，SORL1基因mRNA水平在散发性阿尔茨海默病（SporadicAlzheimer’s disease，SAD）患者以及正常人中存在显著差异。在本次研究中，本课题组通过对SAD患者和健康老年人外周血SORL1基因蛋白编码区（CDS）区进行基因测序分析，发现其常见基因突变位点，并进一步分析SORL1基因突变与其mRNA水平的相关性；同时我们采用硫化测序方式（BS-PCR）以及甲基化特异性PCR（MS-PCR）技术，初步了解SORL1启动子区甲基化状态，为疾病的早期监测提供可能的分子理论依据。第一部分SORL1基因突变和mRNA水平与阿尔茨海默病的相关性研究目的研究散发型阿尔茨海默病患者（SAD）SORL1基因突变与mRNA水平的相关性方法随机选择SAD患者55例（SAD组），另选健康体检者15例（对照组），分别取两组外周血行单个核细胞分离，提取总RNA，反转录合成cDNA，再以cDNA为模板，通过PCR扩增，进行定量PCR检测，最后利用ABI公司分析软件进行定量分析。通过对SORL1基因mRNA水平检测以及全长cDNA测序分析，并将基因突变与未突变SORL1基因mRNA进行水平相关性分析。结果SAD组中有8例出现不同位点的SORL1基因突变，其发生概率为14.5%，较为常见的突变位点为2650以及2850碱基序列，而对照组未检测到SORL1基因突变；SAD组SORL1基因mRNA表达水平较对照组明显减低； SAD组中SORL1基因突变组与未突变组mRNA水平差异无统计学意义（P=0.152）。结论SORL1基因突变参与SAD的发生； SORL1基因突变与SORL1基因mRNA水平无明显相关性。第二部分SORL1基因异常甲基化与阿尔茨海默病关系的研究目的研究SORL1基因启动子区异常甲基化与散发型阿尔茨海默病（SAD）发生的相关性。方法选择散发性阿尔茨海默病（SAD）患者40例为SAD组，另选择健康体检者10例为对照组。提取外周血DNA，，设计硫化测序PCR（BS-PCR）引物以及甲基化特异性PCR（MS-PCR）引物，进行PCR扩增以及测序分析。结果3例健康标本以及3例SAD患者标本DNA经硫化且BSP测序分析，其健康标本甲基化率分别为1.5%、1.0%、1.0%，SAD患者SORL1甲基化率分别为74.0%、70.5%、73.0%；MSP分析结果显示：SORL1基因在10例健康人中呈完全非甲基化状态，在40例SAD患者中其甲基化阳性率为52.5%（p0.05）。结论SORL1基因启动子区异常甲基化可能参与SAD的发生，
[Abstract]:Background Alzheimer's disease (Alzheimer 's disease, AD) is a progressive progressive dementia, which is the most common dementia type. The pathogenesis of 2/3.AD, which accounts for Alzheimer's disease, is related to genetic factors, mainly involved in cell apoptosis, cell differentiation, cell cycle and so on. It is generally believed that the key link of the disease is the key link of the disease. The first is the abnormal accumulation of beta amyloid in the brain neurons and the formation of "senile plaque" and "neurofibrillary tangles". In recent years, many genes related to the brain have been studied. It is considered that the molecular biology techniques are used to analyze the genetic variation and expression of the gene, so that the disease can be diagnosed early by the phenotype change.
The sorting protein related receptor L1 (sortilin-related receptor1gene, SORL1) is one of the new genes associated with the risk of Alzheimer's disease. It is expressed in the nerve cells, and is used as a sorting protein receptor to shuttle in the membrane, cytoplasm and Golgi body, and to participate in the transport and exchange of nerve cells. The expression of mononuclear glycyrrhizic acid in the gene sequence was studied, but the expression of SORL1 gene in peripheral blood was less, especially in the study of mutation and abnormal methylation and late onset Alzheimer's disease. The previous study in our group proved that the level of SORL1 gene mRNA was in sporadic Alzheimer's disease (Spor There are significant differences in adicAlzheimer 's disease, SAD) and normal people. In this study, we found the common gene mutation sites in the SAD patients and the SORL1 gene protein coding region (CDS) region of the peripheral blood of the healthy elderly, and further analyzed the phase of the SORL1 gene mutation and the level of mRNA. At the same time, we use BS-PCR and methylation specific PCR (MS-PCR) technology to understand the methylation status of the promoter region of SORL1, and provide the possible molecular basis for the early monitoring of the disease.
Part one the correlation between SORL1 gene mutation and mRNA level and Alzheimer's disease
Objective to study the correlation between SORL1 gene mutation and mRNA level in patients with sporadic Alzheimer's disease (SAD).
Methods 55 patients with SAD (group SAD) were selected randomly, and 15 cases (control group) were selected for physical examination. Two groups of peripheral blood mononuclear cells were isolated, total RNA was extracted, cDNA was synthesized by reverse transcription, and cDNA was used as a template. Quantitative PCR detection was carried out by PCR amplification. Finally, the quantitative analysis was made using ABI company analysis software. MRNA water of SORL1 gene was used. Flat test and full-length cDNA sequencing analysis, and gene mutation and non mutation SORL1 gene mRNA level correlation analysis.
Results there were 8 cases of SORL1 gene mutation in the SAD group, the occurrence probability was 14.5%, the common mutation site was 2650 and the 2850 base sequence, while the control group did not detect the SORL1 gene mutation, and the mRNA expression level of the SORL1 gene in the SAD group was significantly lower than that in the control group; the SORL1 gene mutation group and the unmutated group mRNA water in the SAD group were in the SAD group. The difference was not statistically significant (P=0.152).
Conclusion the mutation of SORL1 gene is involved in the occurrence of SAD; there is no significant correlation between SORL1 gene mutation and SORL1 gene mRNA level.
The second part is the relationship between abnormal methylation of SORL1 gene and Alzheimer's disease.
Objective to study the correlation between aberrant methylation of SORL1 gene promoter and the occurrence of sporadic Alzheimer's disease (SAD).
Methods 40 patients with sporadic Alzheimer's disease (SAD) were selected as group SAD, and 10 healthy subjects were selected as control group. DNA was extracted from peripheral blood, PCR (BS-PCR) primers were sequenced and PCR (MS-PCR) primers were primed by methylation specific PCR (MS-PCR). PCR amplification and sequencing analysis were carried out.
Results 3 healthy specimens and 3 SAD patients were analyzed by DNA and BSP sequencing. The methylation rates of the healthy specimens were 1.5%, 1% and 1% respectively. The SORL1 methylation rates of the patients with SAD were 74%, 70.5%, 73% respectively. The results of MSP analysis showed that the SORL1 gene was completely methylation in 10 healthy people and the methylation in 40 SAD patients. The positive rate was 52.5% (P0.05).
Conclusion aberrant methylation of SORL1 gene may be involved in the pathogenesis of SAD.

【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

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