臺灣博碩士論文加值系統

摘要
p53抑癌基因是轉錄因子，可藉由活化下游基因，造成細胞生長停止或細胞死亡。因此，找尋p53之下游基因有助於了解p53之作用機轉。本實驗室曾利用mRNA差異表現法由具溫度調控之p53 mutant (tsp53Val135) 之轉型株1-5中，找到可受p53調控之基因DDA1。經由基因庫資料的比對分析，結果找到最近所發現的人類thiamine transporter (hTHTR-1)基因與mDDA1在cDNA及蛋白序列上分別有73%及90%的相同性，顯示mDDA1是human thiamine transporter基因的老鼠同源基因。 為了進一步研究DDA1的subcellular localization及功能，我們建立了以tet-on系統調控的DDA1表現穩定細胞株clone 118，以2 mg/ml Doxycycline處理clone 118細胞24小時後，即可誘發DDA1 mRNA的表現。進一步利用Immunofluorescence以DDA1抗體來偵測，並用confocal顯微鏡觀察，可在加入Doxycycline處理細胞後，清楚的看見DDA1蛋白的表現位於細胞膜上。MTT assay顯示經Doxycycline處理的clone 118細胞，其細胞生長會被部份抑制。
另一方面將clone 118進行thiamine uptake實驗，結果證實在加入Doxycycline後，可看到clone 118細胞對於thiamine uptake的能力比起沒有送入mDDA1的對照組，高出了兩倍左右。證實mDDA1的確具有thiamine uptake的能力。
將293、293T細胞分別以Adriamycin誘發DNA damage之後利用Northern blot觀察，結果在293細胞中（具有wild type p53） hTHTR-1的mRNA增加了，但是在293T（含SV40 large T antigen）細胞中，卻無法看到hTHTR-1 mRNA有增加的情形。初步證實hTHTR-1亦受到p53的調控。

Abstract
p53 tumor suppressor is a transcription factor that causes cell growth arrest and induces apoptosis. Identification of the p53 downstream target genes is therefore important to unravel the mechanisms underlying p53 actions. We have previously identified and cloned a p53-regulated gene, DDA1, by the RNA differential display of an IW32 erythroleukemia stable clone (1-5) that contains a temperature-sensitive p53 mutant gene, tsp53val135. Sequence comparison revealed that mDDA1 shares 73% and 90% identity in its nucleotide and protein sequences, respectively, to the newly identified human thiamine transporter gene (hTHTR-1). To further investigate the subcellular localization and function of DDA1, we established DDA1 expressing clones under the control of the tetracycline inducible promoter. After 24 hours treatment of 2 mg /ml Doxycycline, clone 118 transfectant cells could be induced to express DDA1 mRNA and protein. Immunofluorescence analysis indicated that DDA1 was present on the plasma membrane. Growth of DDA1 stable transfectant was partly inhibited in the presence of Doxycycline. Ability of the clone 118 cells to uptake thiamine increased 2-fold in the presence of Doxycycline. These data demonstrated that mDDA1 possesses thiamine transporter activity.
hTHTR-1 mRNA was induced by DNA damage in a p53 dependent manner. Induction was detected in 293 cells expressing endogenous p53, but not in 293T cells whose p53 was inactivated. Together these results indicate that the high affinity thiamine transpoter is a p53 regulated gene.
中文摘要 ..................................................... 1
英文摘要 ..................................................... 2
緒論 ..................................................... 3
實驗材料 ..................................................... 16
實驗方法 ..................................................... 18
實驗結果 ..................................................... 30
實驗討論 ..................................................... 36
參考文獻 ..................................................... 40
附圖 ..................................................... 47

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