- 學位論文
探討BALF1在EB病毒感染的細胞中所扮演的角色與功能
Studies of The Role of BALF1 in Epstein-Barr Virus Infected cells
摘要
EB病毒是人類疱疹病毒的一員。它能夠持續存活於B淋巴細胞，導致B細胞轉形及癌化。近年來在EB病毒基因研究中，發現有一種會出現在潛伏期及溶裂期的早期溶裂型蛋白質，其結構與Bcl-2相似，稱為BALF1。雖然BALF1與Bcl-2有相似的結構，但我們對其在病毒感染細胞中的角色與功能至今仍不清楚。 為了釐清BALF1在感染細胞的過程中所扮演的角色，我們首先探討BALF1是否影響EB病毒的主要基因。結果顯示BALF1在潛伏感染時期，不影響EBNA1基因的表現，但卻能活化LMP1以及抑制BZLF1，這表示BALF1可能具有維持病毒潛伏感染與增加細胞轉形的能力。但一旦進入溶裂期，BALF1則增加BZLF1表現，這又表示BALF1在溶裂期可能有助於病毒子代的產生。由於BALF1在結構上與Bcl-2類似，因此其功能有可能相同。為證明此點，我們以藥物篩選得到BALF1穩定細胞株，並利用這些細胞株進行細胞轉形、裸鼠腫瘤以及細胞存活等試驗。結果顯示，凡具有BALF1的細胞，其能增加細胞轉形，加速腫瘤產生以及增加細胞存活，但並不能加速細胞週期及改變與細胞週期相關蛋白的表現。另外利用傷口癒合及transwell試驗，證明BALF1可能增加細胞遷移能力。最後我們也發現，BALF1也可藉由增加c-Myc表現，降低細胞內DNA修補能力。此結果與Bcl-2功能相似，它們可能都藉由降低DNA修補能力，並形成基因體不穩定，進而走向癌化。不過我們還是需要更多的實驗證據來證明。 從本篇論文中，我們希望獲得BALF1對其宿主及病毒本身調控機制的資訊，並透過此資訊找出宿主與病毒間互動的關係。藉由彼此關係，期望找出癌化的途徑。
並列摘要
Epstein-Barr virus (EBV) is a human herpesvirus, which is persistent in B lymphoid cells and triggers transformation and tumorigenesis of latently infected B cells in vitro. Recently, an early lytic gene, BALF1, which expressed in both latent and lytic stage and was structurally homologous to Bcl-2 protein, was characterized in EBV infected cells. However the role and function of BALF1 in EBV infected cells still remain to be investigated. To clarify the role of BALF1 in the infected cells, we first examined whether BALF1 regulates the expression of EBV genes. Results showed that BALF1 has no effect on EBNA1 gene expression during latent stage infection, but it simultaneously activated the oncogenic LMP1 and inhibited the BZLF1 gene expression. It seems to imply that BALF1 may function on maintenance of EBV latent infection and cell transformation. Unlike EBV latent stage, BALF1 obviously activated the gene expression of BZLF1 while EBV reactivation occurred, possibly leading to virus production. BALF1 is homologous to Bcl-2 oncogene in structure. It will be logical to speculate that the function of BALF1 is similar to Bcl-2. To prove this, some stable BALF1 transfectants were obtained by using neomycin selection. We used these BALF1 transfectants to perform three tumorigenesis assays, including cell transformation, tumor formation in nude mice and cell survival. The results indicated that all the cells with BALF1 not only activated cell transformation but also promote tumor formation in nude mice and cell survival. But it could not change the process of cell cycle and the expression of cell cycle associated proteins, such as cyclin A and cyclin D1. In addition, the results of both wound healing and transwell assays significantly demonstrated that BALF1 has the ability to increase of metastasis. Finally, we also observed that the increase of c-Myc gene expression and suppression of the cell DNA repair after BALF1 expressed in cells. It is notably implicated BALF1 possibly contributed to tumorigenesis by mimicking the same pathway of Bcl-2, such as c-Myc-mediated suppression of cell DNA repair. However, these evidences are not enough to completely clarify the true role of BAlF1 in cells, we still need to accumulate more evidences to confirm. EBV is a DNA tumor virus, which is associated with many tumors. In this thesis, we look forward to understanding the regulation mechanism of BALF1 in infected cells. This regulation mechanism will provide us to further realize the relationship of EBV and its associated tumors. Furthermore, this information will help us to find the best way of the virus-induced tumorigenesis.