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研究生: 馬振漢
Chen-Han Ma
論文名稱: 人類巨細胞病毒調控增生細胞核抗原表現之機制
Regulation of Proliferating Cell Nuclear Antigen (PCNA) by Human Cytomegalovirus (HCMV)
指導教授: 劉銀樟
Yin-Chang Liu
阮麗蓉
Li-Jung Juan
口試委員:
學位類別: 碩士
Master
系所名稱: 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年: 2004
畢業學年度: 92
語文別: 英文
論文頁數: 67
中文關鍵詞: 人類巨細胞病毒增生細胞核抗原聚合酶輔助因子立即早期蛋白質1立即早期蛋白質2
外文關鍵詞: Human cytomegalovirus, HCMV, Proliferating cell nuclear antigen, PCNA, IE1, UL44, IE2
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  • 人類巨細胞病毒是造成免疫不全者感染的主要原因,當被人類巨細胞病毒感染時,細胞內許多參與去氧核醣核苷酸複製的蛋白質會大量的表現。增生細胞核抗原在去氧核醣核苷酸複製過程中扮演著關鍵性的角色,因此為探討細胞在遭受到人類巨細胞病毒感染後,增生細胞核抗原是否也會大量表現?我們觀察被人類巨細胞病毒感染的細胞中增生細胞核抗原的蛋白質、核醣核苷酸及啟動子的活性是否上升。實驗結果發現,在被人類巨細胞病毒感染後,增生細胞核抗原的蛋白質、核醣核苷酸量增加,啟動子的活性也上升,由此可以顯示人類巨細胞病毒可以經由調控基因的轉錄來增加增生細胞核抗原的表現;我們也經由發現病毒的立即早期蛋白質1能夠活化增生細胞核抗原的啟動子而驗證了此項假設。此外我們也發現,在受到人類巨細胞病毒感染後,增生細胞核抗原在細胞核內會呈現顆粒狀,且和病毒聚合酶輔助因子及立即早期蛋白質2位於細胞核內同樣的位置。因此細胞在受到人類巨細胞病毒感染後,增生細胞核抗原的表現會被病毒的立即早期蛋白質1活化,而增生細胞核抗原蛋白質會被帶到病毒的去氧核醣核苷酸複製的位置。由此現象我們提出一個假設,病毒可能是需要增生細胞核抗原參與病毒的去氧核醣核苷酸複製。


    Human cytomegalovirus (HCMV) is the major infectious cause in immunocompromized individuals. The expression of several cellular proteins involved in modulating cellular DNA replication is increased during HCMV infection. Proliferating cell nuclear antigen (PCNA) is a key protein participating in cellular DNA replication. To determine whether PCNA expression is induced by HCMV, we had investigated the protein, mRNA level, and promoter activity of PCNA and its localization in the nucleus during HCMV infection. We found that HCMV stimulates the synthesis of both protein and mRNA of PCNA and the activation of PCNA promoter indicating that PCNA might be induced by HCMV at the transcriptional level. This hypothesis was further supported by the finding that HCMV viral immediate early protein 1 (IE1) significantly activated PCNA promoter activity. Moreover, PCNA exhibited granular structure in the nucleus and was colocalized with viral polymerase accessory factor UL44 and immediate early protein 2 (IE2) when fibroblasts were infected with HCMV. These results indicate that PCNA is specifically activated by HCMV viral protein IE1 and is possibly recruited to the viral replication center. These results suggest that PCNA may be involved in HCMV DNA replication.

    TABLE OF CONTENTS Abstract ----------------------------------------------------------------------------------------------- i 中文摘要 -------------------------------------------------------------------------------------------- ii Acknowledgements -------------------------------------------------------------------------------- iii Table of contents ----------------------------------------------------------------------------------- iv Chapter 1 Introduction ------------------------------------------------------------------------------ 1 1.1 Human cytomegalovirus (HCMV) is clinically important ------------------------- 1 1.2 HCMV growth is regulated by HCMV viral proteins ------------------------------- 2 1.3 Cell cycle is dysregulated by HCMV ------------------------------------------------- 8 1.4 Proliferating cell nuclear antigen (PCNA): a sliding clamp with ring shape ---- 9 1.5 PCNA: a multi-function protein ------------------------------------------------------ 11 1.6 Does HCMV induce PCNA expression? -------------------------------------------- 15 Chapter 2 Materials and Methods --------------------------------------------------------------- 17 Chapter 3 Results ---------------------------------------------------------------------------------- 27 3.1 Up-regulation of PCNA by HCMV infection --------------------------------------- 27 3.2 HCMV IE1 up-regulates PCNA promoter ------------------------------------------ 29 3.3 PCNA is colocalized with HCMV DNA replication center ----------------------- 30 Chapter 4 Discussion ------------------------------------------------------------------------------ 33 References ------------------------------------------------------------------------------------------ 37 Figures ---------------------------------------------------------------------------------------------- 49 Tables ------------------------------------------------------------------------------------------------ 67

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