|Abstract: ||銅 (Copper, Cu) 是地殼中含量最多的金屬元素之一，在工業及日常生活中有相當廣泛的用途，銅在人體是多種酵素的輔因子，亦參與造血功能，故適當攝取銅對生理功能十分重要。然而，銅也是一種對生物有害的環境汙染物，主要來自電鍍、冶煉、廢五金處理等工業汙染及水產養殖用的除藻劑，銅汙染影響最嚴重者為我國西部海岸的「綠牡蠣」。銅的毒性機制與氧化壓力有關，銅離子 Cu2+ 能誘導活性氧物質 (Reactive oxygen species, ROS) 的大量生成，或催化物質的氧化反應，進而干擾細胞的正常運作，使抗氧化系統失衡、細胞受損、酵素活性異常、DNA 斷裂甚至誘導細胞凋亡途徑，銅離子主要對肝臟及腎臟造成毒性，也被指出和阿茲海默症有關，但其詳細機制仍不明瞭，故本研究將以阿茲海默症相關的小鼠海馬迴神經元 (HT-22) 為細胞模式，探討銅離子對腦神經細胞造成的氧化壓力及毒理機制。 研究第一部分探討銅離子之細胞毒性及氧化壓力，先以細胞存活率試驗及乳酸脫氫酶 (Lactate dehydrogenase, LDH) 活性分別為細胞死亡及受損程度指標，發現隨銅濃度上升，細胞存活率呈現穩定的降低。LDH 釋出量亦顯著增加。顯微鏡觀察下同樣發現細胞逐漸萎縮、破碎甚至懸浮。氧化壓力方面，隨著銅離子濃度增加，ROS 及氧化壓力指標總硫醇基含量分別明顯提升及減低。抗氧化酵素超氧歧化酶 (Superoxide dismutase, SOD) 活性則呈現先升後降的趨勢。綜合上述表示銅離子造成 ROS大量增加，細胞內物質氧化及抗氧化系統受抑制，使細胞死亡。 研究第二部分基於上述結果，探討銅離子造成粒線體功能異常和細胞死亡途徑。調控細胞凋亡的鈣離子濃度及粒線體膜電位 (Mitochondrial membrane potential, MMP) 分別顯著提升和降低，表示內質網壓力的提升，進一步影響 MMP 的消散。因為 MMP 和細胞呼吸作用有關，其強弱能影響能量的產出，故 ATP 生成量亦出現顯著下降。細胞死亡途徑方面，發現凋亡細胞的比例明顯較壞死細胞多，且執行凋亡反應的凋亡蛋白酶 (Cysteine dependent aspartate-specific protease-3/7, caspase-3/7) 活性亦有明顯上升，表示銅離子能夠藉由粒線體途徑對神經細胞誘導凋亡作用，在高濃度下亦能造成細胞壞死。|
Copper (Cu) is one kind of the most rich metal in the earth. There are also many applications in industry. Copper is a cofactor for many enzymes involving in hematopoietic function in human body. Proper copper intake is important for physiological functions. However, copper is also a harmful environmental pollutant, mainly from industrial pollution such as electroplating, smelting, scrap metal processing and algaecide for aquaculture. The most famous case affected by copper pollution is the "green oysters" in the west coast of Taiwan, and copper pollution in water can affect the farming. The toxicity mechanism of copper is related to oxidative stress. Cu2+ can induce the formation of large amounts of reactive oxygen species (ROS) or be the catalyst of oxidation, which can interfere with the normal operation of cells, imbalance of antioxidant system, cell damage, abnormal enzyme activity, DNA cleavage and even induction of apoptosis. Copper ion is mainly harmful to the liver and kidneys and is also indicated to be associated with Alzheimer's disease, but the detailed mechanism is still unknown. Therefore, this study will use Alzheimer's disease-related mouse hippocampal neurons (HT-22) to investigate the oxidative stress and toxicological mechanism of copper ion on brain. The chapter 1 of this study intends to investigate the cytotoxicity and oxidative stress induced by copper ion. With the increase of copper ion concentration, the cell survival rate decreased steadily. The release amount of lactate dehydrogenase (LDH) also increased significantly. The cells were also gradually atrophied, broken or even suspended. In terms of oxidative stress, ROS also increases and total thiol content decreases respectively. The antioxidant enzyme superoxide dismutase (SOD) activity shows a trend of increasing first and then decreasing. In summary, copper ion induces a large increase in ROS with neurons, resulting the induction of intracellular substance oxidation, the inhibition of antioxidant system and cells death. Basing on the results above, the chapter 2 of this study investigates the mitochondrial dysfunction and cell death pathway induced by copper ion. Intracellular calcium concentration and mitochondrial membrane potential (MMP), which regulate apoptosis, are significantly increased and decreased respectively, indicating an increase in endoplasmic reticulum stress (ER stress) and further affecting the dissipation of MMP. Because MMP is related to cellular respiration, its strength can affect energy production, so ATP production also decreases significantly. In term of the cell death pathway, the proportion of apoptotic cells is higher than necrotic cells and the cysteine dependent aspartate-specific protease-3/7 (Caspase-3/7) activity was also increased significantly, indicating that copper ion can induce apoptosis by mitochondrial pathway and necrosis at high concentrations in the neurons.