本文選題：阿爾茨海默病 + SORL1基因�。� 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2013年碩士論文

【摘要】：研究背景阿爾茨海默�。ˋlzheimer’s disease,AD）是一種起病隱襲的進(jìn)行性發(fā)展的癡呆，是最常見(jiàn)的癡呆類(lèi)型，約占老年期癡呆的2/3。AD的發(fā)病機(jī)制與遺傳因素有關(guān)，主要涉及到細(xì)胞凋亡、細(xì)胞分化、細(xì)胞周期等。目前普遍認(rèn)為，發(fā)病關(guān)鍵環(huán)節(jié)之一是β淀粉樣蛋白在腦部神經(jīng)元的異常積聚，形成“老年斑塊”和“神經(jīng)元纖維纏結(jié)”。近年研究與大腦相關(guān)的基因很多,認(rèn)為利用分子生物學(xué)技術(shù)，分析基因結(jié)構(gòu)變異和表達(dá)狀態(tài)，從而對(duì)疾病在表型改變以前進(jìn)行早期診斷。 分揀蛋白相關(guān)受體L1基因（sortilin-related receptor1gene，SORL1）是與阿爾茨海默病的患病風(fēng)險(xiǎn)有關(guān)聯(lián)的新發(fā)基因之一，在神經(jīng)細(xì)胞內(nèi)表達(dá)，作為分選蛋白受體穿梭于胞膜、胞質(zhì)和高爾基體，參與神經(jīng)細(xì)胞的物質(zhì)轉(zhuǎn)運(yùn)和交換過(guò)程。目前歐美等國(guó)家通過(guò)該基因序列中的單核甘酸多態(tài)性進(jìn)行對(duì)照研究，但SORL1基因在外周血中的表達(dá)研究較少，尤其是其突變及異常甲基化與晚發(fā)型阿爾茨海默病的相關(guān)性研究甚少。本課題組的前期研究證明，SORL1基因mRNA水平在散發(fā)性阿爾茨海默�。⊿poradicAlzheimer’s disease，SAD）患者以及正常人中存在顯著差異。在本次研究中，本課題組通過(guò)對(duì)SAD患者和健康老年人外周血SORL1基因蛋白編碼區(qū)（CDS）區(qū)進(jìn)行基因測(cè)序分析，發(fā)現(xiàn)其常見(jiàn)基因突變位點(diǎn)，并進(jìn)一步分析SORL1基因突變與其mRNA水平的相關(guān)性；同時(shí)我們采用硫化測(cè)序方式（BS-PCR）以及甲基化特異性PCR（MS-PCR）技術(shù)，初步了解SORL1啟動(dòng)子區(qū)甲基化狀態(tài)，為疾病的早期監(jiān)測(cè)提供可能的分子理論依據(jù)。 第一部分SORL1基因突變和mRNA水平與阿爾茨海默病的相關(guān)性研究 目的研究散發(fā)型阿爾茨海默病患者（SAD）SORL1基因突變與mRNA水平的相關(guān)性 方法隨機(jī)選擇SAD患者55例（SAD組），另選健康體檢者15例（對(duì)照組），分別取兩組外周血行單個(gè)核細(xì)胞分離，提取總RNA，反轉(zhuǎn)錄合成cDNA，再以cDNA為模板，通過(guò)PCR擴(kuò)增，進(jìn)行定量PCR檢測(cè)，最后利用ABI公司分析軟件進(jìn)行定量分析。通過(guò)對(duì)SORL1基因mRNA水平檢測(cè)以及全長(zhǎng)cDNA測(cè)序分析，并將基因突變與未突變SORL1基因mRNA進(jìn)行水平相關(guān)性分析。 結(jié)果SAD組中有8例出現(xiàn)不同位點(diǎn)的SORL1基因突變，其發(fā)生概率為14.5%，較為常見(jiàn)的突變位點(diǎn)為2650以及2850堿基序列，而對(duì)照組未檢測(cè)到SORL1基因突變；SAD組SORL1基因mRNA表達(dá)水平較對(duì)照組明顯減低； SAD組中SORL1基因突變組與未突變組mRNA水平差異無(wú)統(tǒng)計(jì)學(xué)意義（P=0.152）。 結(jié)論SORL1基因突變參與SAD的發(fā)生； SORL1基因突變與SORL1基因mRNA水平無(wú)明顯相關(guān)性。 第二部分SORL1基因異常甲基化與阿爾茨海默病關(guān)系的研究 目的研究SORL1基因啟動(dòng)子區(qū)異常甲基化與散發(fā)型阿爾茨海默�。⊿AD）發(fā)生的相關(guān)性。 方法選擇散發(fā)性阿爾茨海默�。⊿AD）患者40例為SAD組，另選擇健康體檢者10例為對(duì)照組。提取外周血DNA，，設(shè)計(jì)硫化測(cè)序PCR（BS-PCR）引物以及甲基化特異性PCR（MS-PCR）引物，進(jìn)行PCR擴(kuò)增以及測(cè)序分析。 結(jié)果3例健康標(biāo)本以及3例SAD患者標(biāo)本DNA經(jīng)硫化且BSP測(cè)序分析，其健康標(biāo)本甲基化率分別為1.5%、1.0%、1.0%，SAD患者SORL1甲基化率分別為74.0%、70.5%、73.0%；MSP分析結(jié)果顯示：SORL1基因在10例健康人中呈完全非甲基化狀態(tài)，在40例SAD患者中其甲基化陽(yáng)性率為52.5%（p0.05）。 結(jié)論SORL1基因啟動(dòng)子區(qū)異常甲基化可能參與SAD的發(fā)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.

【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R749.16

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 吳琪;Alzheimer病神經(jīng)原纖維纏結(jié)與tau蛋白研究[J];中國(guó)神經(jīng)精神疾病雜志;2000年01期

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