2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659755摘要：目的：本研究之目的有1. 納入臺灣地區心因性猝死症候群之病患，找出國人特有的SCN5A 突變。2. 以分子生物學之方法來選殖野生型SCN5A 以及國人特有之SCN5A 突變。3. 以膜電位嵌定之方式來對野生型SCN5A 及突變型SCN5A 進行電生理性質的研究。4. 以共軛焦顯微鏡、膜蛋白Biotinylation 及免疫沈澱等方法，研究SCN5A 突變對蛋白質分佈及運送的影響。背景：SCN5A 是心臟細胞鈉離子孔道的基因，負責心肌細胞之動作電位，而近來的研究顯示，SCN5A 的突變可以引起Brugada 症候群，long QT 症候群及先天性心臟傳導阻滯，而這些疾病都可能造成病患的猝死。我們過去的初步研究已發現，國人在這方面有自己特有的基因突變，然而這些突變如何造成臨床疾病，抑或其分子機轉為何至今尚無人研究。方法：1. 我們將納入Brugada 症候群，long QT 症候群及先天性心臟傳導阻滯的病患，由週邊血液中抽取白血球內的DNA，再以PCR 的方法，分成35 個片段放大其SCN5A 基因，再以核苷酸定序找出SCN5A 基因的突變。2. 我們將自心臟移植手術摘除的心臟中萃取人類心臟之mRNA，以反轉錄之方式產生cDNA，再以PCR 的方式，放大並選殖SCN5A 及SCN1B 基因，並以site-directed mutagenesis 的方法來得到各種突變的SCN5A 基因。3. 我們將含有SCN5A 及SCN1B 的pBudCE4.1 質粒transfect 進入HEK293 細胞，它們分別是鈉離子孔道的α及β次單位，可以形成完整的鈉離子孔道，再以膜位電位嵌定的形式進行鈉離子孔道的電氣生理性質研究。4. 我們將以共軛焦顯微鏡來研究SCN5A 在細胞內的分佈，首先將SCN5A 基因在選殖過程中與myc motif 作fusion，表達於HEK293 細胞後以anti-myc 抗體來偵測，並以Cy3 之螢光來顯影，細胞背景則以pBudCE4.1 中的綠螢光蛋白顯影，在共軛焦顯微鏡下可直接觀察SCN5A 的分佈。5. 我們將以細胞表面蛋白biotinylation 的方法來偵測SCN5A蛋白是否可以被運送到細胞的表面。將細胞表面蛋白biotinylation 後，接著抽取其蛋白，以anti-myc抗體作免疫沈澱，萃取其SCN5A，再以streptavidin 來偵測這些SCN5A 是否有biotinylation.預期之成果：經由本研究我們預期得到下列的研究成果。1. 我們可以找出國人特有的SCN5A 突變，這不但是重要的本土性資料，而且對產前諮詢及優生學具有重要意義。2. 針對國人特有的SCN5A 突變，我們可以詳細了解其致病之分子機轉。SCN5A可以造成多種不同的心因性猝死症候群，可見其突變具多種不同的功能性障礙，而透過本研究，我們不但能了解國人特有的SCN5A 突變如何致病，並能對了解SCN5A 複雜的功能作進一步的貢獻。<br> Abstract: Specific aims:1. To include patients with hereditary sudden cardiac death syndrome and screen forunique SCN5Amutations.2. To clone wild type SCN5A sodium channel and distinct mutations in Taiwan.3. To investigate the basic electrophysiological properties of wild type and mutantSCN5A sodium channels by performing patch clamp studies.4. To investigate the protein processing and trafficking properties of wild type andmutant SCN5A sodium channels by confocal microscope, surface biotinylation andimmunoprecipitation.Background:SCN5A encodes the cardiac sodium channel and is responsible for the actionpotential in most cardiomyocytes. Mutations of the SCN5A gene have been reported tocause Brugada syndrome, long QT syndrome and congenital cardiac conduction defect,all of which are possible etiologies of sudden cardiac death. Our past researches haveidentified several unique SCN5Amutations in Taiwanese patients. However, themechanisms why these mutations cause clinical ventricular arrhythmia remain unclear.Methods:1. We will include patients with sudden cardiac death syndrome and genomic DNAwill be isolated from leukocytes in peripheral blood. Polymerase chain reaction willbe performed to amplify the SCN5A gene. Direct DNA sequencing will used todetect mutations in these genes2. Human cardiac mRNA will be obtained from explanted human heart. Reversetranscription will be performed to obtain cDNA and the coding region of SCN5Aand SCN1B gene will be amplified using PCR. Mutant clones will be obtained bysite-directed mutagenesis.3. We will express the plasmid pBudCE4.1 containing both SCN5A and SCN1B inHEK293 cells. SCN5A encode the sodium channel a-subunit and SCN1B theb-subunit and they will form a complete cardiac sodium channel. Patch clampstudies will be applied to investigate the basic electrophysiological properties.4. We will use confocal microscopic technique to investigate the distribution ofSCN5A protein in HEK 293 cells. The SCN5A protein will be fused with myc motifand detected by Cy3-conjugated anti-myc antibody. The background fluorescencewill be green fluorescent protein produced by the pBudCe4.1 plasmid.5. Cell surface protein biotinylation studies will be performed to test whether theSCN5A protein reach the cell surface membrane. The cell will be treated withsurface biotinylation agents. The cellular protein will then be collected,immunoprecipitated with anti-myc antibody and treated with strepavidin. OnlySCN5A that reaches the cell surface will be detected..Expected results:1. We expected to detected distinct SCN5Amutations responsible for sudden cardiacdeath syndrome in Taiwan. These are valuable local information and will be veryuseful for genetic counciling and eugenics.2. The pathogenesis of SCN5A is complex and therefore SCN5Amutations may causedifferent syndromes.With our studies, we expect to elucidate the mechanisms of ionchannel defect and contribute to the understanding about the molecular mechanismsof SCN5A-related diseases.心因性猝死症候群鈉離子孔道膜位電位嵌定共軛焦顯微鏡sudden cardiac death syndromecardiac sodium channelpatch clampconfocal microscope