臺灣博碩士論文加值系統

呈等比級數生長期的人類纖維母細胞(Human fibroblast, HF)培養於亞砷酸鈉(sodium arsenite, SA)的培養液中4小時，用SRB方式檢測細胞存活率，其50%的致死濃度介於30μM - 40μM，若濃度超過80μM 時，人類纖維母細胞存活率則低於 20%，表示亞砷酸鈉對人類纖維母細胞具有細胞毒性。
亞砷酸鈉處理會提高細胞培養液中亞硝酸的含量，間接代表一氧化氮(nitric oxide, NO)形成，亞砷酸鈉濃度愈高，產生愈高量一氧化氮，另外，同樣的處理，也會提高細胞內nitrotyrosine 含量代表peroxynitrite(NO與superoxide反應而成的產物)伴隨著一氧化氮生成而增加，此物質會硝化細胞內的蛋白質（造成對蛋白質的破壞）。NAME 3.2mM與亞砷酸鈉10μM 共同處理組，與單獨處理亞砷酸鈉10μM比較，細胞存活率高了15﹪，顯示一氧化氮生成是亞砷酸鈉引起的細胞死亡的原因之一。
以亞砷酸鈉處理人類纖維母細胞2小時或4小時後，麩胱甘還原酵素(glutathione reductase, GR)活性隨亞砷酸鈉的濃度以及處理時間增加而降低，4小時亞砷酸鈉40μM處理下，麩胱甘還原酵素活性只剩57%，亞砷酸鈉濃度大於80μM時，無論4小時或2小時處理，麩胱甘還原酵素活性皆低於50%。以4小時亞砷酸鈉處理後，繼續無藥培養12小時及24小時，觀察麩胱甘還原酵素活性回復程度，亞砷酸鈉40μM，12小時與24小時，麩胱甘還原酵素活性回升20 - 30%，亞砷酸鈉濃度大於120μM時，後培養24小時僅回復30%，結果並顯示，人類纖維母細胞在24小時內無法100﹪恢復麩胱甘還原酵素活性。分別用亞砷酸鈉40μM，亞砷酸鈉40μM與NAME1.6mM，亞砷酸鈉40μM與NAME 3.2mM處理人類纖維母細胞4小時後測量麩胱甘還原酵素活性，亞砷酸鈉與NAME 1.6mM共同處理，與單獨處理亞砷酸鈉比較，麩胱甘還原酵素活性回升 4% - 10%，亞砷酸鈉加NAME 3.2mM則回升 4% - 30%，表示麩胱甘還原酵素活性因亞砷酸鈉所引發的NO被抑制。
人類纖維母細胞經亞砷酸鈉4小時培養後，麩胱甘(glutathione, GSH)含量呈現出下降的趨勢。亞砷酸鈉 40μM處理組之GSH降低了16%，亞砷酸鈉160μM，其含量只剩44%。 與GSH對照的是GSSG含量，和控制組相比，亞砷酸鈉40μM處理組 已增至2.6倍，細胞內的GSH總量(GSH與GSSG含量相加)，則沒有明顯的變化 --指出GSH的合成在4小時培養時間內，無顯著改變。麩胱甘還原酵素與亞砷酸鈉 10μM共同處理組，和單獨處理亞砷酸鈉10μM比較，細胞存活率高20﹪，顯示麩胱甘還原酵素活性的降低是亞砷酸鈉引起的細胞死亡的原因之一。

Human fibroblasts were exposed to sodium arsenite for 4h. The cytotoxicity of sodium arsenite treatment was determinted by SRB assay. The IC50 was 30μM- 40μM. Here, the cell survival was only 20﹪of control in the exposure of 80μM sodium arsnite. This study has shown that sodium arsnite influence the cytotoxicity of HF.
Treatment of HF with SA40-160μM for 4h increase the nitrite content released into cell cultured medium,indicating the elevated level of nitric oxide (NO). Peroxynitrite is a powerful modifier of intracellular proteins. The nitration of tyrosine , nitrotyrosine , is a useful marker for detecting peroxynitrite in biological samples.HF cells exposure to SA40-160μM for 4h increase nitrotyrosine level by 10-20﹪,indicating the elevated level of peroxynitrite. Cotreament with 3.2mM Nω-nitro-L-arginine methyl ester ( NAME) and 10μM SA for 4h, the survival rose from 65﹪to 78﹪. These data suggest that NO was involed in SA-induced cell death.
HF cells incubated with 40-160μM SA showed glutathione reductase activity significantly decrease to less than 29-60﹪after 2h and less than 43-65﹪after 4h. After HF cells were exposed to 40μM SA for 4h and followed a postincubation in drug-free medium for 12 or 24h. Glutathione reductase(GR) activity rose from 20﹪to 30﹪after 12 and 24 h of recovery. However,GR activity did not recover to the control level wthin 24h. Cotreament of HF cells with SA and NAME（1.6 or 3.2mM） for 4h , partly restore the GR activity. At 1.6mM NAME,the percentage of the recovered GR activity was 4﹪-10﹪.At 3.2mM NAME,the percentage of the recovered GR activity was 4﹪-30﹪. The data showed that treatment with SA increased nitrite production in HF cells and that NAME could not significantly recover GR activity.
HF cells exposure to SA40-160μM for 4h decrease GSH level by 16-56nmol /mg protein. This decrease in GSH content was accompanied by a simultaneous increase in GSSG level, with a GSH disappearance and GSSG production.4h after an incubation with 40μM SA, GSSG increase about 2.6-fold.Intracellular total glutathione concentration were almost unchanged suggesting that GSH synthesis did not differ from control cells after 4h. Cotreament with GR and 10μM SA for 4h, the survival rose from 65﹪to 82﹪. These data suggest that decreased glutathione reductase induced by sodium arsenite, lead to cell loss of viability.

目錄
中文摘要 1
英文摘要 3
縮 寫 5
緒 論 6
實驗方法 14
結 果 21
討 論 26
參考文獻 33
圖 表 43

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