Abstract
細胞質麩胱甘生過氧化酵素 ( glutathione peroxidase )是一種胞質抗氧化酵素，以 GSH 提供氫離子還原 H2O2 ( hydrogen peroxidase ) 和有機過氧化物 ( organic hydroperoxidase )。過去之研究證實：將 GPx酵素缺失的 xrs-5 細胞，以牛的綜合 GPx 酵素處理，能降低短時間處理亞砷酸納所誘導的微小核球生成。並且，在 CHO-K1 細胞中，加入 GPx酵素的抑制劑，會增加短時間處理亞砷酸納所引發的微小核球生成。然而，細胞中 GPx-1 在亞砷酸鈉與 H2O2 誘發的氧化逆境下所扮演的角色並未完全了解。 為了解亞砷酸納與 H2O2 處理後的 HF 細胞 GPx-1 表現的程度，本研究首先製備合適的人類 GPx-1 蛋白質作為抗原，並製備抗體作為識別細胞中 GPx-1 蛋白質表現的探針 （ probe )，以檢查處理後細胞 GPx-1 蛋白質表現。在 gpx-1 基因選殖方式是由人類纖維母細胞的mRNA 為材料，以 RT-PCR 製備 GPx-1 cDNA，所得 GPx-1 cDNA 連接pQE32 載體後，轉殖入 SG1009 E.coli。選取有 gpx-1基因表現的菌株，製備大量的 GPx-1 蛋白質作為抗原。然後，以 GPx-1 抗原多次注射兔子後，得到 GPx-1 的抗體血清。抗體血清的測試則以稀釋 3000 倍後，利用西方轉漬法檢查，結果可偵測到大於 12.5 ng 的 GPx-1 抗原。並且，以相同的稀釋倍數也能偵測到 50 μg 以上人類纖維母細胞萃取物中的GPx-1 蛋白質。 應用稀釋 3000倍的抗體血清為探針（ probe ）以西方轉漬法檢查：人類纖維母細胞處理亞砷酸納與 H2O2 後，GPx-1 蛋白質表現，結果顯示：處理亞砷酸納 ( 0 ~ 400μM ) 4 小時或 H2O2（ 0 ~160μM）30 分鐘後，GPx-1 蛋白質增加 1 到 3.3 倍。處理亞砷酸納 (2.5 ~ 5μM ) 24 小時後，蛋白質增加到 2~3.4 倍。然而，在處理亞砷酸納 ( 10 ~ 20μM ) 24 小時， GPx-1 並沒有被偵測到。以相同條件下處理人類纖維母細胞後，應用 RT-PCR 方式分析 gpx-1 mRNA 表現則與蛋白質表現不同。結果顯示：亞砷酸納 4 小時處理組，隨劑量增加，gpx-1mRNA 表現提高 ( 從 1 增加到 1.9 倍）。處理亞砷酸納 24 小時後，各處理組之 gpx-1 mRNA 的轉譯，均比未處理組減少，約為其 0.4 倍。然而，在處理 H2O2 後之各組 gpx-1 mRNA 的轉譯程度，與未處理組幾乎相同。Cellular glutathione peroxidase ( GSH-Px1 ) is a nuclearencoded cytosolic antioxidant enzyme that reduces hydrogenperoxide and organic hydroperoxides with GSH as the hydrogendonor. However, it was found that the addition of commercialbovine glutathione peroxidase ( GPxs ) would decrease arsenite-induced micronucleus ( MN ) formation or genotoxicity in GPxdeficient xrs-5 cells. And, the GPx inhibitor increase MNformation in CHO-K1 cells. To further determine whether GPx-1plays a role to protect HF cells from arsenite-induced oxidativestress, GPx-1 antibodies were produced and used as probe forexamining GPx-1 protein. The gpx-1 cloning, GPx-1 expression,GPx-1 antibodies production and GPx-1 antibodies titration areas followings: human GPx1-cDNA was subcloned into commercialyavailable pQE32 expression vector ( Quiagen ) and transfectedinto SG1009 E. coli. The transformed clone having the highestGPx expression was used to produce large quantity of GPx-1protein as antigen. Antibodies to GPx-1 were produced fromserum after several injections of GPx-1 antigen into rabbits.The results in Western blotting analysis showed that 1:3000dilution of antibodies-containing serum could detect 12.5 ngGPx-1 antigen. In addition, GPx-1 in about 50 μg of humanfibroblast extract could be detected using same titration.To examine GPx-1 expression in human fibroblasts after arseniteor H2O2 treatment, GPx-1 antibodies of 3000-fild dilution andwestern blotting were used. The results showed a dose-dependentincrease ( form 1 to 3.3-fold ) of GPx-1 expression in cellsafter treatment with 0-400μM aresenite for 4h or 0-160μM H2O2for 30 min. More ( 2~3.4-fold ) GPx-1 expression is found incells after treatment with 2.5-5μM aresenite for 24h than thosein untreated control. However, no GPx-1 expression was detectin cells treated with 10 or 20μM aresenite for 24h. Appliedreverse transcription-coupled PCR analysis to examine gpx-1 mRNAin treated human fibroblasts, the results did not correlate withthose of GPx-1 protein expression after same treatmentprotocols. The results showed a dose-dependent increase ( form1 to 1.9-fold ) of GPx-1 mRNA transcription in cells aftertreatment with 0-400μM aresenite for 4h. Less ( 0.4-fold only) GPx-1 mRNA transcription from cells treated with aresenite for24h at above concentration ( 0-20μM ) were found than thosefrom untreated control. In addition, no difference in GPx-1mRNA transcription between sample from 0-160μM H2O2 treatmentand untreated control.