銅離子為生物體內維持正常生理恆定功能的重要微量金屬元素，參與許多細胞內的生化反應。然而，過量之銅離子在體內堆積亦會造成某些生理功能異常。為此在人類細胞體內，主要靠兩套運輸銅離子恆定的金屬穿膜蛋白，分別為ATP7A及ATP7B。在斑馬魚 (Danio rerio)，相對於ATP7A的同源基因，其主要功能為運輸細胞內銅離子至TGN (trans-Golgi network)，提供銅離子給需銅酵素，以執行正常生理功能。為了了解ATP7A參與銅離子代謝中的角色，我們利用合成的寡聚核酸互補的序列 (Morpholino) 抑制ATP7A基因表現，再透過微陣列資料分析斑馬魚胚胎基因變化，由此發現某些基因表現量受到影響，其中包含SOD1 (Cu/Zn superoxide dismutase)基因。SOD1是體內重要的抗氧化酵素，根據先前之研究，已知銅離子會影響SOD1的酵素活性表現。然而，SOD1如何受銅離子及ATP7A調控其基因轉錄則仍不清楚。因此我們藉由製造銅離子缺乏、或提供不同銅離子的斑馬魚胚胎生長環境，來改變細胞內銅離子恆定，以定量PCR偵測斑馬魚胚胎SOD1基因表現變化，以釐清ATP7A如何透過銅離子影響SOD1基因的表現。我們的結果也發現，SP1可能參與調控SOD1轉錄，因而ATP7A可能透過SP1路徑及銅離子的存在，來調控SOD1基因表現。
The trace element copper plays important roles in a range of physiological functions. However, copper is also toxic to cell once when the cation built up. The dilemma is unraveled by two similar mechanisms to carefully control the uptake and rejection of the cation to maintain a copper homeostasis. In human, two genes are responsible for the homeostasis, ATP7A and ATP7B. In zebrafish (Danio rerio), the ortholog of the ATP7A, which is required for copper transport from its intracellular storage to trans-Golgi network (TGN) in order to be used by cuproenzyme or for cellular efflux. To evaluate the role of ATP7A in copper metabolism, an antisense morpholino oligonucleotide strategy was performed to knockdown the zebrafish ATP7A. Using microarray to analyze the ATP7A knockdown zebrafish mRNA, some genes are found up- or down-regulated by the treatment. A variety of cuproenzyme were affected including the superoxide dismutase (SOD1), a significant component of antioxidant system. Although it was known that cofactor copper is required for SOD1 enzyme activity, there is no evidence to reveal the possibility that ATP7A regulates the SOD1 gene expression. To evaluate the role of copper in SOD1 gene regulation, zebrafish embryos were knockdown their ATP7A gene or applied to a copper deficient (by copper chelator) or overdosed environments. Our results have shown that transcription factor SP1 is important for SOD1 expression. Based on these data, a mechanism that ATP7A down-regulates SOD1 gene expression is through SP1 mediated by copper ions is established.
