突變型超氧化物岐化酵素Cu,Zn-SOD引發神經細胞死亡誘發運動神經元疾病ALS之分子致病機制探討

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Title:	突變型超氧化物岐化酵素Cu,Zn-SOD引發神經細胞死亡誘發運動神經元疾病ALS之分子致病機制探討 The Molecular Action Mechanism of Human Cu,Zn-SOD Gene Deficiency-induced Cell Death in Amylotrophic Lateral Sclerosis
Authors:	陳奕平 Yi-Ping Chen
Contributors:	醫學研究所
Keywords:	突變型超氧化物岐化酵素運動神經元疾病神經細胞 Cu Zn-SOD ALS
Date:	2001
Issue Date:	2009-09-11 15:30:43 (UTC+8)
Abstract:	Amyotrophic Lateral Sclerosis (ALS) 是一種進行性運動神經肌肉萎縮症，屬於運動神經元疾病(MND)的一種，目前病因不明，臨床上尚無有效治療藥物。而在FALS病人的sod1基因上，已發現約有50種點突變。推測ALS病因可能是:由於人體內sod1基因發生點突變使體內產生過量的自由基hydroxyl radical (‧OH)無法獲得清除，進而攻擊神經細胞造成細胞走向apoptosis死亡。apoptosis是造成ALS病人運動神經受傷萎縮的主因。因此我們探討突變型sod1基因引起神經細胞死亡誘發運動神經元疾病ALS之致病機制。為進一步篩檢台灣本土型 FALS病例，探討台灣本土型 FALS病例是否與日本及歐美的病例一般發生sod1基因點突變，設計了5對PCR引子，深入分析台灣本土型 FALS病例 sod1基因所發生變異的位置。利用RT-PCR的技術從人類肝臟組織cDNA中選殖出存在人類體內的抗氧化酵素: Cu,Zn-SOD並以大腸桿菌E. coli進行蛋白質基因工程的表現及其多株抗體的製備，成功的選殖出抗氧化酵素基因，表現出具有活性約1335 (Unit/mg) 的蛋白質。此外利用site-direct mutagenesis PCR構築出三個未曾分析過帶有點突變的sod1基因E21K、D90V和D101G，選擇大腸菌基因表現系統製備出這些變異型重組蛋白質，結果顯示他們的活性下降分別只剩至52%、56%、38%，在in vitro上相似於病人來證明其體內酵素活性的改變與差異。 PC12細胞為神經疾病方面良好的細胞實驗模式，為探討變異的sod1基因與 ALS 相關性，目前我們成功新建立了SOD1-transduction delivery protein系統，超越傳統transfection技術的缺點,可以定量將正常或變異型的蛋白質直接準確的送入PC 12 細胞。以免疫螢光染色法定性觀察到denature Tat-SOD1明顯被攜帶入細胞，而且隨著時間的增加SOD1蛋白的存在量也跟著增加，更重要的是也觀察到酵素活性的增加發揮所扮演的生理活性功能。進一步探討神經細胞透過apoptosis的死亡機制，在細胞培養液中給與一個已知的superoxide anion產生物70mM Paraquat，使其對PC12細胞產生攻擊引起死亡。細胞存活度約為18%。隨著Tat-SOD1、Tat-E21K和D101G不同蛋白的加入，他們的存活度分別增加為33% (Tat-SOD1)、29% (Tat-E21K)，在DNA fragmentation中細胞所出現的斷裂情形有明顯差異，顯示他們可以保護細胞免於走向死亡;然而加入Tat-D101G細胞的存活度卻下降為6%，出現特別顯著促進細胞死亡的情形。隨著人類基因的解碼及後基因體時代的來臨，我們更希望藉由transduction delivery protein系統進行研究,相信必定可以達到protein therapy的目標。 The most frequent genetic causes of amylotrophic lateral sclerosis (ALS) determined so far are mutations occurring in the gene coding for copper/zinc superoxide dismutase (Cu, Zn-SOD).The mechanism may involve the formation of hydroxyl radicals or malfunctioning of the SOD protein. We found that sequence of genomic sod1 gene from a sporadic ALS patient has two point mutations.In order to investigate the possible roles on genetic causes of ALS, SOD cDNA and mutants were constructed and transduced into PC12 cell to observe the cellular consequences. RT-PCR from human liver extract generated the SOD gene (sod1). Wild-type sod1 was constructed into a transcription-translation expression vector to examine the SOD1 production in vitro. Wild-type SOD1 was highly expressed in Sf-9 cells infected by Baculovirus and in E. coli. Active SOD1 was expressed by IPTG induction and in a metal-dependent matter.SDS-PAGE and Western blot analysis illustrated a 23kDa monomer product.To study the cellular effect of SOD1, Tat-tag was used as a cellular transduction delivery vehicle. Denatured Tat-sod1 was observed to be successfully transduced into undifferentiated PC12 or differentiated PC12 neural cells and retained its activity via protein refolding. Site-directed mutagenesis created a genetic deficiency in tat-tag wild-type with three point mutations as E21K, D90V and D101G. Tat-tag mutants expressed in E. coli completely lost SOD activity.In undifferentiated PC12 cells, Tat-SOD avoided DNA fragmentation from superoxide attack generated by 70mM paraquat; however, mutant Tat-D101G enhanced cell death.
Data Type:	thesis
Appears in Collections:	[Graduate Institute of Medical Sciences] Dissertation/Thesis

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