摘要
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。ALS可分為兩型 : 85%~90%屬於偶發性( Sporadic, SALS ) ; 其他15%~10% 屬於遺傳性 ( Familial, FALS )。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，無法清除體內所產生過量的自由基hydroxyl radical (?OH )，進而攻擊神經細胞造成細胞走向apoptosis死亡。Apoptosis被認為是造成ALS病人運動神經受傷萎縮的主因，因此我們探討突變型sod1基因與神經細胞死亡而誘發的運動神經元疾病ALS之致病機轉的關係。 HIV-1 病毒具有Tat蛋白，此Tat蛋白與HIV-1的複製有關。Tat蛋白主要是由4個domain所組成，其中以basic domain最重要，使病毒的Genome DNA具有轉位的能力。目前的研究已知Tat蛋白可攜帶其他的蛋白質，穿透細胞膜進入細胞內。 PC12細胞是一研究神經疾病良好的細胞實驗模式，可用來探討變異的sod1基因與 ALS 之間的相關性。因此我們以未分化的PC12 cell為細胞實驗模式進行protein ( Tat-SOD1 fusion protein ) transduction的實驗。目前已成功的建立了protein transduction delivery system，超越傳統transfection技術的缺點。 我的主要論文內容是以正常小鼠為動物實驗模式，研究Tat-SOD1 融合蛋白是否可以經由腹腔注射或藉由其他的方式進入老鼠體內，穿透細胞膜及血腦障壁，到達腦部及各個組織器官的細胞內，並觀察其分佈情形。
並列摘要
The most frequent genetic causes of amylotrophic scelosis (ALS) determined so far are mutations occurring in the gene coding for copper/znic superoxide dismutase (Cu,Zn-SOD). The mechanism may involve the formation of hydroxyl radicals or malfunctioning of the SOD protein . The development of peptide drugs and therapeutic proteins is limited by the pool permeability and the selectivity of the cell membrane. A series of small protein domains , termed protein transduction domains (PTDs) , have been shown to cross biological membranes efficiently and independently of specific transporters and receptors , and to promote the delivery of peptides and proteins into cells. The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which posses a characteristic positive charge on the basis of their enrichment of arginine and lysine residues activates transcription by specifically binding a stem-loop element in the viral long terminal repeat and is responsible for highly efficient protein transduction through the plasma membrane. Denatured Tat-SOD1 fusion protein was observed to successfully transduced into undifferentiated PC12 cells and retained its activity via protein refolding. It had been demonstracted that intraperitoneal injection of Tat-β -galactosidase fusion protein resulted in delivery of the biologically active fusion protein to all tissues in mice, including the brain (Dowdy, 1999). According to it, we also purify the Tat-SOD1 fusion protein and want to observe that Tat-SOD1 fusion protein also can delivery efficiently to all tissue in mice by intraperitoneal injection.
參考文獻
延伸閱讀
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