本文選題：阿爾茨海默病　切入點(diǎn)：presenilin-1基因　出處：《浙江大學(xué)》2012年碩士論文

【摘要】：研究背景： 阿爾茨海默病(Alzheimer's disease, AD)是一種進(jìn)行性不可逆神經(jīng)系統(tǒng)變性疾病,是老年癡呆最常見(jiàn)的病因。至今AD的確切病因和發(fā)病機(jī)制不明,目前已知載脂蛋白Eε4等位基因與遲發(fā)性AD相關(guān),早發(fā)性家族性AD主要由β-淀粉樣前體蛋白(P-amyloid precursor protein, βAPP)、早老素-1(presenilin-1, PS-1)及早老素-2(presenilin-2, PS-2)基因突變引起。目前對(duì)于PS-1基因突變?nèi)绾沃虏∵�不清楚,之前的研究認(rèn)為突變的PS-1通過(guò)“獲得功能”(gain of function)影響β-淀粉樣多肽1-42(Amyloid β1-42,Aβ42)而發(fā)病,即淀粉樣蛋白級(jí)聯(lián)假說(shuō)(Amyloid Cascade Hypothesis),該假說(shuō)認(rèn)為異常的β淀粉樣蛋白(amyloid-P peptides, Aβ)是觸發(fā)病理級(jí)聯(lián)反應(yīng)并最終導(dǎo)致AD的第一步。Ap是由正常存在于細(xì)胞膜上的APP經(jīng)p-和γ-分泌酶剪切后產(chǎn)生一系列長(zhǎng)度在39～43個(gè)氨基酸的短肽,其中,Aβ42最容易聚集纖維化,是主要的致病蛋白。早老素(presenilin, PS)蛋白是γ-分泌酶復(fù)合物的活性亞基,如發(fā)生突變,可影響APP剪切而導(dǎo)致異常Aβ的產(chǎn)生和聚集。近期的研究發(fā)現(xiàn),某些PS-1基因突變不通過(guò)Aβ42致病。因此,需要更多的研究參與闡述PS-1基因突變與AD的關(guān)系。 目的： 通過(guò)篩查突變頻率最高的PS-1基因突變來(lái)觀察111例來(lái)自浙江大學(xué)醫(yī)學(xué)院附屬第二醫(yī)院神經(jīng)內(nèi)科住院或門診被診斷為AD的患者并且未經(jīng)家族史和發(fā)病年齡篩選的情況下PS-1基因的突變情況。篩查發(fā)現(xiàn)1例患者PS-1基因3號(hào)外顯子起始密碼子發(fā)生了雜合突變ATG→GTG,構(gòu)建突變型質(zhì)粒轉(zhuǎn)染細(xì)胞觀察PS-1起始密碼子突變后PS-1和APP的表達(dá)變化情況。 方法： 研究對(duì)象共266例包括未經(jīng)過(guò)家族史和發(fā)病年齡篩選的111例來(lái)自浙江大學(xué)醫(yī)學(xué)院附屬第二醫(yī)院神經(jīng)內(nèi)科住院或門診被診斷為AD的患者,5例M766患者親屬,150例正常對(duì)照。常規(guī)酚/氯仿提取法提取外周血DNA,對(duì)PS-1基因13個(gè)外顯子及側(cè)翼內(nèi)含子DNA擴(kuò)增,聚合酶鏈反應(yīng)(polymerase chain reaction, PCR)產(chǎn)物純化后直接測(cè)序分析。利用體外定點(diǎn)突變技術(shù)突變野生型PS-1cDNA并構(gòu)建野生型和突變型PS-1增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein, EGFP)融合基因質(zhì)粒,通過(guò)實(shí)時(shí)熒光定量PCR檢測(cè)PS-1和APP基因在人胚胎腎(HEK)293細(xì)胞、小鼠成神經(jīng)瘤細(xì)胞N2a中的mRNA表達(dá)情況,并通過(guò)Western blot技術(shù)觀察野生型和突變型PS-1蛋白在兩個(gè)細(xì)胞系中的表達(dá)變化情況。 結(jié)果： (1)發(fā)現(xiàn)患者M(jìn)766的PS-1基因3號(hào)外顯子起始密碼子發(fā)生了雜合突變ATG→GTG。該患者的發(fā)病年齡為57歲。其93歲母親、63歲的大妹妹和患者的小兒子也檢測(cè)到同樣的突變,但均未發(fā)病。同時(shí),發(fā)現(xiàn)3個(gè)已報(bào)道的單核苷酸多態(tài)(Single Nucleotide Polymorphisms, SNP)(rs1800839,rs116882898,rs165932)。 (2)實(shí)時(shí)熒光定量PCR檢測(cè)發(fā)現(xiàn)PS-1基因mRNA在HEK293細(xì)胞和N2a細(xì)胞中野生型均顯著高于突變型,APP基因mRNA在兩細(xì)胞系中未見(jiàn)明顯差異。 (3)Western blot結(jié)果顯示野生型和突變型PS-1在HEK293細(xì)胞中均表達(dá),且突變型表達(dá)量較野生型少。而N2a細(xì)胞僅表達(dá)野生型PS-1。 結(jié)論： (1)對(duì)未經(jīng)過(guò)家族史和發(fā)病年齡篩選的111例來(lái)自浙江大學(xué)醫(yī)學(xué)院附屬第二醫(yī)院神經(jīng)內(nèi)科住院或門診被診斷為AD的患者行PS-1基因突變篩查未發(fā)現(xiàn)新的或已經(jīng)報(bào)道的致病突變,提示PS-1基因突變導(dǎo)致的AD占總AD患者比例較低。 (2)根據(jù)M766患者家系圖顯示,未見(jiàn)PS-1基因起始密碼子突變和疾病AD共分離現(xiàn)象,推測(cè)其可能不是致病突變,而是AD的一個(gè)危險(xiǎn)因素,也可能是一個(gè)SNP。 (3)實(shí)時(shí)熒光定量PCR檢測(cè)發(fā)現(xiàn)PS-1基因mRNA在HEK293細(xì)胞和N2a細(xì)胞中野生型均顯著高于突變型,Western blot技術(shù)檢測(cè)PS-1蛋白表達(dá)顯示野生型和突變型PS-1在HEK293細(xì)胞中均表達(dá),且突變型表達(dá)量較野生型少。而N2a細(xì)胞僅表達(dá)野生型PS-1,提示PS-1基因非ATG起始密碼子GTG在HEK293細(xì)胞中表達(dá)效率降低,而在神經(jīng)樣細(xì)胞N2a中可能引起PS-1蛋白不表達(dá)。 (4)實(shí)時(shí)熒光定量PCR檢測(cè)發(fā)現(xiàn)APP基因mRNA在兩細(xì)胞系中未見(jiàn)明顯差異,提示PS-1基因起始密碼子突變對(duì)APP影響不大。
[Abstract]:Research background:
Alzheimer's disease (Alzheimer's disease AD) is a kind of irreversible neurodegenerative disease is the most common cause of senile dementia. The exact etiology and pathogenesis of AD is unknown, currently known apolipoprotein E 4 allele associated with late-onset AD, early-onset familial AD is mainly composed of beta - amyloid precursor protein (P-amyloid precursor protein, P APP), presenilin -1 (presenilin-1, PS-1) -2 early old (presenilin-2, PS-2) caused by gene mutation. The mutation of PS-1 gene to pathogenesis is unclear, the research before that mutant PS-1 by "function" (gain of function) effects of beta amyloid polypeptide 1-42 (Amyloid beta 1-42, beta 42 A) and the incidence, namely the amyloid cascade hypothesis (Amyloid Cascade Hypothesis), the hypothesis that amyloid beta protein abnormalities (amyloid-P peptides, A beta) is the pathological cascade trigger counter The first step should be.Ap and eventually AD is normally present in the cell membrane of APP by p- and gamma secretase produces a series of length 39~43 amino acid peptide, the A beta 42 most likely to gather the fibrosis is the main pathogenic protein. Presenilin (presenilin, PS) protein is the active subunit of the gamma secretase complex, such as mutation of APP can affect the shear caused by abnormal aggregation and A beta. A recent study showed that some mutations in the PS-1 gene by A beta 42 pathogenesis. Therefore, more research is needed in this relationship between PS-1 gene mutation and AD.
Objective:
By screening the mutation of PS-1 gene in the highest frequency of mutation was observed in 111 patients from the Second Affiliated Hospital of Zhejiang University Department of Neurology inpatient or outpatient PS-1 gene was diagnosed with AD and without family history and age of onset of the absence of screening the mutation screening. 1 cases were found in exon 3 of PS-1 gene start codon occurrence the heterozygous mutation of ATG, GTG, construction of mutant plasmid transfected cells to observe the expression changes of PS-1 and APP mutation after PS-1 initiation codon.
Method錛，

本文編號(hào)：1705544