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研究生(外文):Mao-Wen Weng
論文名稱(外文):Expression of MDM2 mRNA splicing variants during tumorigenesis
指導教授(外文):Jiunn-Liang Ko
外文關鍵詞:tumor suppressor gene-p53oncogene-mdm2
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MDM2為人類的致癌基因,在一些腫瘤組織中經常可以發現MDM2基因的異常表現。而台灣肺癌中也發現,男性MDM2發生缺失的比例較女性高,同時在透過對肺癌組織的分析,經由篩選後選用6種不同短小缺失的MDM2,依其長度命名,分別為MDM2-1181、MDM2-1026、MDM2-1025、MDM2-891、MDM2-657、MDM2-259,並利用基因選殖的方式將上述短小缺失的MDM2與表現載體pCDNA3.0或pCDNA-HA進行接合反應,再利用DNA轉染的方式和MDM2 p53-dependent promoter一起表現於H1299/p53wt肺癌細胞株中,觀察這些短小缺失的MDM2在細胞中調控p53的情形,來分析其對於肺癌細胞可能造成的影響。結果顯示MDM2-891的luciferase表現情形與MDM2-1476相差無幾,而其它形式的MDM2則普遍低下。再進一步轉染MDM2-891、MDM2-1476及p19ARF質體DNA至H1299/p53wt肺癌細胞中並以西方轉漬法去分析p53wt表現的情形,結果顯示在單獨轉染MDM2-1476的情況下,細胞內p53蛋白明顯的減少;而在單獨轉染MDM2-891的情況下,p53的量並無明顯減少;接下來更進一步選用MDM2-657、MDM2-891與MDM2-1476質體DNA,並利用in vitro translation方式得到其所轉譯出的蛋白,其蛋白大小依序為 24.4 kDa、24 kDa與55.2 kDa,再與使用大腸桿菌所表現純化而得到的p53蛋白去進行免疫沉澱分析,發現三者對於p53蛋白均有結合反應,顯示在此兩類缺失型的胺基酸序列中尚有一未發現的部位仍具有與p53結合的能力。

The mdm2 is an oncogene that is activated by overexpression in several human tumors. In Taiwan, some distinct MDM2 mRNA transcripts including full-length RNA, splicing MDM2 forms, were detected in lung carcinomas by Nested-PCR and the ratio of MDM2 deletion mutants in male were higher than in female. To understand the biological function of the splicing MDM2 forms that may play the role in tumorigenesis, We had also cloned several splicing MDM2 protein translations(MDM2-1181,MDM2-1026,MDM2-1025,MDM2-891,MDM2-657,MDM2-259), and H1299/p53wt cell were transfected with these splicing MDM2 plasmids and MDM2 p53-dependent promoter. We analyzed the characterization with its ability to regulate p53 via luciferase activity assay. The result show, MDM2-891 exhibited the luciferase activity similar with MDM2-1476, but others was not. We present evidence to show if the MDM2-1476 and MDM2-891 expressed from their respective cDNA could degrade the wt p53 . H1299/p53wt cells were transfected with MDM2-1476, MDM2-891 and p19ARF plasmids and analyzed the expression of p53 protein by Western Blotting. These result show, p53 protein was degraded by MDM2-1476, but not degraded by MDM2-891. We analyzed the molecular weight of MDM2 trunscated forms by in vitro translation and use the products to immunoprecipitate with p53 protein which is amplified from E.coli expression system and purified from Glutathione-Sepharose 4B affinity column. Our findings indicated the presence of the splicing MDM2 forms which could bind to p53 protein. It showed that MDM2-891 and MDM2-657 might keep a serious of unknow sequences which could bind to p53 protein. These observations suggest that MDM2 expression is altered in lung carcinomas and the cellular roles of these trunscated MDM2 isoforms remains not to be clarified.

二、中文摘要--------------------------------------------------------------- 5
三、英文摘要--------------------------------------------------------------- 7
四、緒言--------------------------------------------------------------------- 9
五、研究動機--------------------------------------------------------------- 22
六、實驗材料與儀器------------------------------------------------------ 25
七、實驗方法--------------------------------------------------------------- 28
八、實驗結果--------------------------------------------------------------- 51
九、討論--------------------------------------------------------------------- 58
十、圖表及圖表說明------------------------------------------------------ 67
十一、參考文獻--------------------------------------------------------------- 82

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