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標題: | 雙特異性去磷酸酶2及6在EB病毒感染細胞中所扮演之角色 Role of Dual-Specificity Phosphatase 2 and 6 in Epstein-Barr Virus Infected Cells |
作者: | Yu-Hsin Lin 林宇馨 |
指導教授: | 蔡錦華(Ching-Hwa Tsai) |
關鍵字: | EB 病毒,MAPKs,雙特異去磷酸酶,Zta, EBV,MAPKs,DUSPs,Zta, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | EB 病毒(Epstein-Barr virus)為雙股 DNA 病毒,主要感染 B 細胞與上皮細胞。EB 病毒感染和淋巴球增生疾病及人類惡性腫瘤形成有高度相關性。被感染的 B細胞會不朽化(immortalize)成具持續增生能力之淋巴母芽細胞株(lymphoblastoid cell line, LCL) ,並誘發許多訊息傳遞路徑如 MAPK 的活化。為瞭解 EB 病毒感染是否會透過調控雙特異性去磷酸酶 (Dual-specificity phosphatases, DUSPs)而影響MAPK 訊息傳遞路徑,並影響細胞增生與凋亡,特此探討該蛋白家族中 DUSP2與 DUSP6 在 EB 病毒感染細胞中所扮演之角色。
實驗室先前以 cDNA 微陣列分析 CD19+ B 細胞及以 B95.8 strain EB 病毒轉形而成之 LCLs,發現 DUSP1、DUSP2、DUSP6 及 DUSP8 在 LCLs 中表現量均較未感染之 B 細胞低。進一步以 RT -Q-PCR 比較 EB 病毒感染後不同時間點DUSPs 家族 mRNA 表現情況,發現隨著 EB 病毒感染時間增長,大多數 DUSPs基因表現均有被抑制的現象。 本篇研究發現在巴氏淋巴瘤、霍金氏淋巴瘤及上皮細胞株中,DUSP2 及 DUSP6 的 mRNA 及蛋白表現均受到 EB 病毒轉錄活化子 Zta 抑制。利用螢光酵 素報導基因分析則發現 Zta 會抑制 DUSP2 及 DUSP6 的啟動子活性。分析 Zta 蛋白結構可發現 Zta 的 DNA 結合域是影響 DUSP2 啟動子活性的重要區域。分析DUSP2 及 DUSP6 的啟動子則發現 Zta 可能不會直接與 DUSP2 啟動子結合而直接影響其轉錄;但 Zta 會透過 DUSP6 啟動子-1003/-546 區間序列影響 DUSP6 轉錄。 在 LCLs 中大量表現 DUSP2 或 DUSP6,則發現 MAPKs 之磷酸化會受到抑 制,且會抑制細胞的增生。顯示 EB 病毒可能藉由抑制 DUSPs 表現而影響細胞 生理功能,然而其他更詳盡的調控機制仍有待進一步研究探討。 Epstein-Barr virus (EBV) is a double stranded DNA virus that mainly infects B cells and epithelial cells. EBV infection is highly correlated with a variety of lymphoproliferative diseases and human carcinomas. EBV can immortalize primary B cells into proliferated lymphoblastoid cell lines (LCLs), and induce various signaling cascades, including MAPK pathway. In order to investigate whether EBV viral products activate the MAPK signals through regulating the Dual-specificity phosphatases (DUSPs) expression and affecting cell proliferation or apoptosis, this study focus on the role of DUSP2 and DUSP6 of DUSPs family protein in EBV infected cells. Previously, we used cDNA microarray to compare the expression pattern of cellular genes in CD19+ B cells with B95.8 strain EBV -transformed LCLs, and found that the gene expression of DUSP1, DUSP2, DUSP6, and DUSP8 are lower in LCLs than those in uninfected CD19+ B cells. Further study by RT -Q-PCR showed that the mRNA expressions of most DUSPs family are down-regulated through the EBV infection. This study showed that DUSP2 and DUSP6 are down-regulated by EBV transactivator Zta at mRNA and protein level in Burkitts’ lymphoma cell line, Hodgkin lymphoma cell line, and epithelial cells. By performing luciferase reporter assay, we demonstrated that Zta can regulate the expression of DUSP2 and DUSP6 through inhibiting its promoter activity. Analyzing the protein structure of Zta, we found that the DNA binding domain of Zta might be important for repression of DUSP2 promoter activity. By analyzing the promoter sequence of DUSP2 and DUSP6, we found that Zta may not bind to DUSP2 promoter directly to prevent DUSP2 from transcription. However, Zta can repress DUSP6 expression through the DUSP6 promoter region between -1003 and -546 site. Further study showed that exogenous expression of DUSP2 and DUSP6 result in down-regulation of MAPKs phosphorylation and cell proliferation in LCLs. Suggesting EBV viral products affect the biological function of the cells through repressing the expression of DUSPs. However, further investigations will be needed to dissect the detail regulatory mechanisms between EBV and DUSPs. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61748 |
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顯示於系所單位: | 微生物學科所 |
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