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研究生:強伍翎
研究生(外文):Wu-Lin Charng
論文名稱:D型肝炎病毒大、小抗原蛋白對細胞內轉錄因子的不同影響
論文名稱(外文):Large and Small Hepatitis Delta Antigens Have Differential Effects on Cellular Transcription Factors
指導教授:吳妍華
指導教授(外文):Yan-Hwa Wu Lee
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:80
中文關鍵詞:D型肝炎
外文關鍵詞:HDV
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D型肝炎病毒(HDV)需要B型肝炎病毒(HBV)提供外套膜蛋白才能包裹成完整的病毒顆粒,為一缺陷型病毒。D型肝炎病毒的大、小型D型肝炎病毒抗原蛋白,在病毒生活史中扮演不同的角色。小型D型肝炎病毒抗原蛋白對於病毒的複製是必須的;相反的,大型D型肝炎病毒抗原蛋白,則會抑制病毒的複製,促進病毒顆粒的包裹。

本論文旨在探討大、小D型肝炎病毒抗原蛋白分別在D型肝炎病毒感染中所扮演的角色。由於很多探討D型肝炎病毒抗原蛋白對宿主細胞影響的研究是從轉錄的層面開始,因此在本文中大規模地篩選受D型肝炎病毒抗原蛋白影響的轉錄因子。首先,建構可以持續地分別表現大型與小型D型肝炎病毒抗原蛋白的HuH-7細胞株。以TranSignalTM Protein/DNA arrays分析這些細胞株中哪些轉錄因子的DNA結合強度受到HDAg的影響。實驗發現受到大型D型肝炎病毒抗原蛋白影響的轉錄因子大部分的DNA結合強度是增強的,然而大部分受到小型D型肝炎病毒抗原蛋白影響的轉錄因子DNA結合強度是變弱的。挑出八種與肝炎、肝硬化,以及肝癌相關的轉錄因子,如:HNF-4, Smad3/4, Stat1, Stat3, IRF-1/2, Oct-1, E4BP4 and NF-�羠,進行電泳遷移率試驗(EMSA)。除了HNF-4與Smad3/4的DNA結合能力不受大、小型D型肝炎病毒抗原蛋白影響之外,其他轉錄因子的結果與TranSignalTM Protein/DNA arrays的結果是吻合的。

以luciferase reporter assay進一步地探討這些DNA結合能力的改變反映到轉錄活性的情形。出人意料地,大型D型肝炎病毒抗原蛋白不影響NF-�羠的artificial reporter,而小型D型肝炎病毒抗原蛋白對此reporter有些微的抑制作用。此外,大型D型肝炎病毒抗原蛋白對於Stat的artificial reporter有dose-dependent增強效果,而小型D型肝炎病毒抗原蛋白卻沒有這樣的影響,與array data的結果是一致的;當轉染顯性抑制 (dominant negative) 的Stat3到細胞中,大型D型肝炎病毒抗原蛋白的活化能力就受抑制消失了。以西方墨點法發現Stat3 的DNA結合能力增加,有一部份原因來自於Stat3從細胞質進到細胞核。D型肝炎病毒對宿主細胞的病理影響可能來自於大、小型D型肝炎病毒抗原蛋白直接或間接影響細胞中轉錄因子所致。
Hepatitis delta virus (HDV) is a defective virus, which requires hepatitis B virus (HBV) to supply surface antigens (HBsAg) for maturation and secretion. In addition, the small hepatitis delta antigen (SHDAg) and large hepatitis delta antigen (LHDAg) of HDV have differential roles in HDV life cycle. Specifically, SHDAg promotes HDV RNA replication and LHDAg inhibits replication but promotes virion assembly.

In this study, we investigated the role of LHDAg and SHDAg in the pathogenesis of HDV infection, respectively. First of all, LHDAg and SHDAg permanent HuH-7 cell lines were generated and cellular transcription factors affected by HDAg are screened with TranSignalTM Protein/DNA arrays. The binding intensity of most transcription factors influenced by LHDAg was enhanced; on the contrary, SHDAg has suppressive effects on the binding intensity of most of these transcription factors. The variation in binding intensity of several pathogenesis-related transcription factors, such as HNF-4, Smad3/4, Stat1, Stat3, IRF-1/2, Oct-1, E4BP4 and NF-�羠 were further confirmed with the electrophoresis mobility shift assays (EMSA). Only the binding intensity of HNF-4 and Smad3/4 were not affected by LHDAg or SHDAg, the others approximately consistent with the array data.

The transactivity of these deregulated transcription factors were investigated via the luciferase reporter assay. Surprisingly, SHDAg, but not LHDAg, had only slightly inhibitory effect on NF-�羠-Luc. On the other hand, LHDAg but not SHDAg transactivated APRE-Luc (acute phase response elements) in a dosage-dependent manner. Further transfection with the dominant negative HA-Stat3F suppressed such activation. The enhancement of binding intensity of Stat3 might be partially due to the translocation. HDAg may have their pathogenetical effect from deregulation of certain transcription factors directly or indirectly.
Chinese Abstract………………………………………………………1
English Abstract……………………………………………………….3
Introduction……………………………………………………………5
Discovery and Classification of Hepatitis D Virus……………………………..5
The Life Cycle of HDV…………………………………………………………...5
The Assembly of HDV Viral particle…………………………………………….6
The Functional Domains of HDAg……………………………………………….7
The Modification of HDAg………………………………………………………10
Cellular Targets of HDAg………………………………………………………..12
Material and Methods…………………………………………………16
Materials……………………………………………………………………………..16
Escherichia coli strain……………………………………………………………16
Cell line……………………………………………………………………………16
Plate and medium………………………………………………………………...16
Plasmids…………………………………………………………………………...16
Solution……………………………………………………………………………19
Chemicals…………………………………………………………………………21
Enzymes……………………………………………………………………….….21
Primer…………………………..…………………………………………….…..21
Isotope…………………………………………………………………………….21
Antibodies………………………………………………………………………...21
Methods……………………………………………………………………………...22
Competent Cell Preparation and Transformation……………………………..22
Plasmid Purification……………………………………………………………...22
Cell Culture……………………………………………………………………….22
Transient Transfection…………………………………………………………...22
Permanent Cell Lines Generation………………………………………………23
Fractionation……………………………………………………………………..23
Western Blotting………………………………………………………………….24
TranSignalTM Protein/DNA Arrays……………………………………………...24
Electrophoresis Mobility Shift Assay (EMSA)………………………………….25
Luciferase Assay………………………………………………………………….26
Results…………………………………………………………………..27
Generation of LHDAg and SHDAg permanent cell lines……………………...27
The Effect of HDAg on the DNA Binding Intensity of Transcription Factors..27
The Effect of LHDAg and SHDAg on Stat3 and Stat1…………………………28
The Effect of LHDAg and SHDAg on NF-B…………………………………..30
The Effect of LHDAg and SHDAg on Oct-1……………………………………30
The Effect of LHDAg and SHDAg on E4BP4…………………………………..31
The Effect of LHDAg and SHDAg on Other Transcription Factors………….32
Discussion………………………………………………………………34
Reference……………………………………………………………….39
Tables and Figures……………………………………………………..47
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