系統識別號 U0026-0812200910212625
論文名稱(中文) p53於Saos-2細胞中對於rat Mrp3基因轉錄調控之研究
論文名稱(英文) Transcriptional regulation of multidrug resistance-associated protein 3 (Mrp3) gene expression by p53 in Saos-2 cells
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 90
學期 2
出版年 91
研究生(中文) 吳筱珈
研究生(英文) Hsiao-Chia Wu
學號 s2689402
學位類別 碩士
語文別 中文
論文頁數 90頁
口試委員 指導教授-黃金鼎
中文關鍵字 多重抗藥性相關性蛋白  轉錄調控 
英文關鍵字 Sp1  p53  Mrp3  transcriptional regulation  multidrug resistance-associated protein 
中文摘要 中文摘要
多重抗藥性相關性蛋白(Multidrug resistance-associated protein, MRP)為ATP binding cassette轉送器超級家族中,重要的轉送器之一。而目前發現的多重抗藥性相關性蛋白家族,是由MRP1~MRP7所組成,其分別也都在體內扮演著不同的生理角色。MRP3在體內為一有機陰離子的轉送器,目前MRP3也被認為是體內膽鹽的腸肝循環中扮演重要角色的轉送器之一。在1992年的文獻指出,MDR基因啟動區的活性表現會受到p53的調控。當轉染野生型p53時會抑制MDR啟動區的活性,而轉染突變型p53時,反而會抑制野生型p53對MDR啟動區的調控。1998年的文獻亦指出,MRP1基因的表現會受到轉錄因子 p53及Sp1的調控,Sp1會增強MRP1 基因啟動區的活性,而野生型p53會抑制Sp1所刺激增加的MRP1基因啟動區的活性。在我們實驗室已發表的文獻中指出,rat Mrp3基因啟動區上有五個Sp1 binding site (-436~440,-213~-219,-107~-118,-69~-74)。我們將rat Mrp3基因啟動區做 5’端不同長度deletion,並在其後接上luciferase reporter gene,同時將其與利用tetracycline off系統調控之野生型p53於Saos2細胞進行轉染,找出p53最主要影響在rat Mrp3 基因啟動區-157~-19的位置。而先前我們實驗室發表的文獻已指出,在-157~-19 啟動區上的第三與第四個Sp1 binding site對於rat Mrp3 基因啟動區的活性扮演著重要角色,但在我們的實驗結果中發現野生型p53並非藉由第四與第五個Sp1 binding site來抑制rat Mrp3基因啟動區的活性,若將第三個Sp1 binding site前的半個p53 binding site 去除也不影響野生型p53抑制rat Mrp3 基因啟動區活性。而當野生型p53表現會抑制rat Mrp3基因啟動區活性表現時,一些癌症中常見的突變型p53 (V143A, R248Q, R249S, R283P )卻無法抑制rat Mrp3基因啟動區的活性表現。因此,依據我們的實驗結果目前已知p53確實可調控Mrp3 基因啟動區的活性,但其機轉並非藉由結合於第四或第五個Sp1 binding site或第三個Sp1 binding site前的半個p53 binding site來進行其抑制作用。
英文摘要 Abstrate
Multidrug resistance-associated protein (MRP) is an important subfamily of the ATP-binding cassate superfamily. Seven MRP isoforms (MRP1~MRP7) have been identified in humans and have been shown to exhibit a tissue-specific distribution and play different physiological functions. MRP3 is an organic anion transporter in the normal physiological condition and it may play an important role in enterohepatic circulation of the bile salt. It was reported that MDR gene promoter is regulated by p53. The MDR promoter activity is down regulated by co-transfected with wild type p53, but when co-transfected with mutant type p53, the MDR promoter activity which is down regulated by wild type p53 can be reversed. In another report, it has been found that Sp1 can upregulate the MRP1 promoter activity, but wild type p53 can repress the activity which is stimulated by Sp1. Our previous studies indicated that there are five Sp1 binding sites (-436~440,-213~-219,-107~-118,-69~-74) in the rat Mrp3 promoter region. We used the 5’ deletion method to get different length of the rat Mrp3 promoter, and then constructed the different rat Mrp3 promoter fragment to the pGL3-basic plasmid adjacent to the luciferase reporter gene. Then, the tetracycline off system was used to express p53 or not. When co-transfect rat Mrp3 promoter reporter gene and p53 in Saos-2 TetOff cells, we found that wild-type p53 inhibited the rat Mrp3 promoter activity at the major region of –157~-19. In our previous studies indicated that the third and fourth Sp1 binding site (-107~-118) play important role in rat Mrp3 promoter activity, but in this study we find the wild type p53 does not inhibit rat Mrp3 promoter activity through the fourth or the fifth Sp1 binding site, or through the half p53 binding in front of the third Sp1 binding site. And four mutant type p53 (V143A, R248Q, R249S, R283P) were found to lose the ability to inhibit the rat Mrp3 promoter activity. In our study, we consider that rat Mrp3 gene expression is regulated by p53, and tumor-derived mutant p53 mutated in the p53 DNA binding domain abolish the p53 repression activity of Mrp3.
論文目次 目錄
第一章 緒論…………………………………………...……………………..6
第二章 實驗材料……………………………………………..…………….15
第三章 實驗方法……………………………………………………..…….24
第四章 實驗結果
第一節 探討p53是否調控Mrp3基因啟動區的表現………...…….………49
第二節 探討野生型p53抑制Mrp3基因啟動區之機轉……………....…….53
第三節 探討突變型p53是否調控Mrp3基因啟動區的表現………………..56
第四節 探討野生型p53對於老鼠迴腸細胞IEC-18中內生性Mrp3 mRNA表現之影響58
第五章 總結與討論60
第六章 參考文獻66
參考文獻 第六章 參考文獻
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