近年來許多學者的研究，猜測三價砷所造成的毒性，是與三價砷會產生自由基有關。在細胞中為了抵抗許多的氧化壓力，細胞自己便會發展許多防禦的機制。Thioredoxin peroxidase II (TPX II) 為一個新發現的抗氧化蛋白質，且被發現廣泛的存在於許多不同的種類中。 在本篇論文中，我們研究在哺乳細胞中的TPX II會受到亞砷酸鈉的調節且TPX II會與亞砷酸鈉鍵結。先前我們實驗室已經證實，TPX II蛋白會過量表現於中國倉鼠卵巢抗砷細胞株SA7中(SA7細胞是持續培養在含有亞砷酸鈉的培養液中)。在本研究中，利用定量聚合酶連鎖反應及西方點墨法，探討亞砷酸鈉影響TPX II基因及蛋白表現的情形。由結果發現，三價砷確實會誘發TPX II基因及蛋白的表現，且呈現時間相關性。為了更進一步確定，在亞砷酸鈉所誘發的毒性中，TPX II所具有的生理功能，將TPX II 轉植株細胞 (KB-TPX及3T3-TPX)處理亞砷酸鈉 (Sodium arsenite)和過氧化氫 (H2O2)。結果發現，轉植株細胞KB-TPX較能抵抗過氧化氫的毒性，而對亞砷酸鈉的毒性則較無抗性。最近的結果亦顯示，在大腸桿菌系統中表現的TPX II蛋白和SA7細胞中的TPX II蛋白，能直接與亞砷酸鈉產生鍵結。亞砷酸鈉已被提及對蛋白質的硫醇基有高度親和性。因此目前利用在TPX II蛋白的半胱胺酸進行點突變，以期能更進一步確定三價砷與TPX II蛋白鍵結的主要胺基酸位置。 Recently, numerous studies suggested that arsenite-induced toxicity is associated with the generation of free radicals. To cope with the oxidative stress, the cell has developed various defense mechanism. A novel antioxidant protein, thioredoxin peroxidase II (TPX II), was identified in a wide variety of species. In this thesis, we investigated the involvement of TPX II in arsenite-mediated toxcity in mammalian cells, and the binding of TPX II with sodium arsenite. Previously our laboratory has identified that TPX II protein was overexpressed in SA7 cells (an arsenic resistant cell routinely cultured in arsenite-containing medium). In this study, the effect of As3+ on the TPX II gene and protein expression were further explored by quantitative PCR and Western blotting analysis. The results showed that arsenite induced TPX II gene and protein expression in a time-dependent manner. To identify the physiological function of TPX II in arsenite-induced toxicity, the TPX II-transfected KB (KB-TPX) and 3T3 (3T3-TPX) cells were treated with sodium arsenite and hydrogen peroxide (H2O2). The results showed that KB-TPX cells exhibits more resistant to H2O2, but not sodium arsenite. The current results also show that sodium arsenite bind directly to TPX II in E.coli expression system and in SA7 cells. Arsenite has been proposed with high affinity for sulfhydryl groups of protein. Therefore, site-directed mutagenesis in cysteine residues of TPX II are currently underway to further identify the binding site of TPX II with arsenite.
