鎘、砷、鉛、汞為生物體非必需重金屬元素，攝取量過多時會對生物體造成嚴重毒害。文獻指出，鎘會影響細胞骨架中肌動蛋白絲的聚合，而肌動蛋白絲的聚合會受到切絲蛋白的調控。在先前實驗室的研究中發現，鎘的刺激會藉由PI3K/Rac1/PKD1/SSH1L途徑造成切絲蛋白活化。因此，本論文試圖了解砷、鉛、汞在HEK293細胞中，是否對切絲蛋白活性產生影響及其相關訊號傳遞。研究中發現處理砷、鉛、汞後，切絲蛋白活性僅受砷調控活性上升，並發現PTEN在細胞中含量隨砷劑量上升而下降。PTEN在細胞中負責PI3K的負調控，因此我們觀察砷誘導切絲蛋白是否透過PTEN/PI3K。在細胞中表現PTEN或處理PI3K抑制劑，都無法影響砷處理下切絲蛋白活性。分析下游因子發現Rac1、RhoA及PKD1在砷誘導下，切絲蛋白活性皆不受影響。文獻指出Ras/Raf/MEK透過p21抑制ROCK來調控切絲蛋白。在砷刺激下發現ERK活性上升，但處理MEK抑制劑發現，切絲蛋白活性不受影響，並且發現細胞中p21蛋白質量不受砷影響，然而ROCK mRNA含量受砷刺激而下降。切絲蛋白活性主要受到LIMK及SSH1調控。處理砷後發現，SSH1L活性上升，LIMK1蛋白質含量下降。結果發現砷會透過ROCK/LIMK及SSH1L共同調控切絲蛋白活性。

Cadmium, arsenic, lead, and mercury are non-essential elements for plant or animal life. Excessive uptake may cause severe toxicological effect to organisms. Previous studies indicated that cadmium affects the polymerization of actin cytoskeleton. And the polymerization of actin filament is regulated by cofilin activity. Previous study from our laboratory indicated that cadmium treatment activates cofilin activity via PI3K/Rac1/PKD1/SSH1L cascade. We investigated in this study the effects of arsenite, lead, and mercury on cofilin activity and the involved signaling pathway in HEK293 cells. We found that the treatment of arsenite increased cofilin activity and decreased PTEN protein level. However, overexpression of PTEN did not affect arsenite-induced cofilin activation. PTEN is a negative regulator of PI3K pathway. Addition of PI3K inhibitor had also no effect on cofilin activity. Further study of downstream effectors indicated that Rac1, RhoA, and PKD1 was not involved in the activation pathway. Reportedly, cofilin activity modulates by ROCK through Ras/Raf/MEK/p21 pathway. We found that ERK would be activated in the treatment of arsenite. However, addition of MEK inhibitor did not affect arsenite-induced cofilin activity. We also found that p21 protein level in cells was not altered by arsenite but mRNA level of ROCK was decrease. On the other hand, cofilin activity is modulated mainly by LIMK and SSH1. We found that arsenite treatment increased SSH1L activity but decreased LIMK1 protein level. Our results indicated that ROCK/LIMK and SSH1L coordinately regulate arsenite-induced cofilin activation.

謝誌 2
目錄 3
中文摘要 4
英文摘要 5
緒論 6
材料與方法 15
結果 22
討論 32
參考資料 39
附圖 47

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