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研究生:陳信旭
研究生(外文):CHEN HSIN-HSU
論文名稱:在鼻咽癌細胞中,分析EBV一特別在表皮細胞使用之啟動子ED-L1E的轉錄調控複合體
論文名稱(外文):Analysis of Transcription Regulation Complex of An Epithelial Cell-Specific EBV Promoter ED-L1E In NPC Cells
指導教授:張玉生張玉生引用關係
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:40
外文關鍵詞:EBVLMP1Sp1HLTF
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LMP1基因的表現可從ED-L1 (EBV基因體nt 169571-nt 169498)和 ED-L1E (nt 170159-nt 170132)兩個啟動子開始;ED-L1可以轉錄出2.8 kb訊息RNA,ED-L1E可以轉錄出3.5 kb訊息RNA。ED-L1啟動子主要是在B淋巴球和表皮細胞中會使用,而ED-L1E則在表皮細胞具功能。因此我們假設在ED-LIE上有一特別會在表皮細胞中形成的轉錄調控複合體。在之前的研究已知此啟動子會被特別表現在表皮細胞中的Sp1和Sp3調控,但是否尚有其它的細胞分子在此複合物則仍為未知數。我利用ED-L1E啟動子DNA,與細胞核萃取物進行結合反應,再以質譜儀鑑定到一個Helicase like transcription factor (HLTF)。此外,我也將ED-L1E的片段分成4個區域以biotin標定(分別為probe 1, probe 2, probe 3,及probe 4),與NPC-TW02細胞核萃取液進行EMSA實驗,其中probe 1與核萃取液形成一個DNA-蛋白質複合體,當分別以Sp1及HLTF之抗體進行supershift,而兩種抗體則沒有造成明顯的supershift,未來仍需重複實驗進一步證實。
Expression of LMP1 gene can be initiated from two promoters: ED-L1 (EBV genome nt 169571- nt 169498) and ED-L1E (nt 170159- nt 170132). Two transcripts with molecular sizes of 2.8 kb and 3.5 kb are transcripted by ED-L1 and ED-L1E, respectively. ED-L1 is used in both B lymphocytes and epithelial cells, whereas ED-L1E is preferentially used in epithelial cells. Thus, we hypothesize that the transcription regulation complex that determines the usage of ED-L1E can be specifically formed in epithelial cells. Previous study suggests that ED-L1E can be regulated by cellular transcription factors Sp1 and Sp3 which are ubiquitously expressed in epithelial cells. However, it is not clear what other cellular factors may be involved in forming this transcription complex that can regulate ED-L1E activity. Using the ED-L1E DNA to incubate with the nuclear extracts of NPC-TW02 cells, the proteins potentially contained in the complex were pulled down. After analyzed on 8-15% gradient SDS-PAGE, followed by identified through MALDI-TOF analysis, Helicase like transcription factor (HLTF) was identified. By EMSA study, I further showed that probe 1 (nt 170165 - nt 170135) of ED-L1E could form a complex with nuclear extracts. This DNA-protein complex had no obvious supershift, when anti-Sp1, anti-HLTF or both antibodies were added. Further validation of the data is merited.
指導教授推薦書.......................................................
口試委員會審定書…………………………………………
授權書……………………………………………………iii
Acknowledgements………………………………………… iv
Chinese abstract………………………………………………v
Abstract...................................... vi
Contents…………………………………………………vii
Introduction………………………………………………1
Materials and methods…………………………………………7
Results……………………………………………………15
Discussion………………………………………………18
References………………………………………………20
Figures……………………………………………………25
Tables……………………………………………………39
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