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標題: | 探討CD300a在受EB病毒不朽化之類淋巴母芽細胞株中的表現及調控 Expression and regulation of CD300a in Epstein-Barr virus-immortalized lymphoblastoid cell lines |
作者: | 楊宜瑾 Yi-Chin Yang |
指導教授: | 蔡錦華 Ching-Hwa Tsai |
關鍵字: | EB病毒,CD300a,EBNA2,Zp, EBV,CD300a,EBNA2,Zp, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | EB病毒(Epstein-Barr virus, EBV)是一具有致癌性的病毒,跟許多的人類疾病有高度的相關性,包含了巴氏淋巴瘤 (Burkitt’s lymphoma)、霍杰金氏症 (Hodgkin’s disease) 、鼻咽癌( Nasopharyngeal Carcinoma, NPC) 、移植後淋巴組織增生症(Post-transplant lymphoproliferative disorder, PTLD)、瀰漫型大B 細胞淋巴癌(Diffuse large B cell lymphoma, DLBCL)等。在體外系統中,EB病毒可感染人類B細胞,使其不朽化成具持續增生能力的類淋巴母芽細胞株(lymphoblastoid cell line, LCL),而LCL的基因表現模式跟PTLD有非常多相似性,因此LCL可作為在體外系統中研究EB病毒致癌機轉的模型。
實驗室先前利用cDNA微陣列分析CD19+初級B細胞及LCLs基因表現的變化情形,如預期地,LCLs與初級B細胞的基因表現呈現了許多不同,而在其中,一分化簇標記分子—CD300a的表現量在LCLs中顯著的升高。並且CD300a表現量上升的情形只在EB病毒感染時發生,而非利用其他刺激物活化B細胞時,如anti-CD40/ IL4, poly I:C, 或LPS。由於CD300a本質是一抑制性受體,在免疫細胞中調控許多免疫反應。因此本研究欲探討在EB病毒感染B細胞後,是如何調控CD300a以及CD300a在其中扮演的生理功能。 實驗結果顯示,在EB病毒感染B細胞後,會透過EBNA2 (EBV nuclear antigen 2)誘發CD300a的表現。根據螢光酵素報導分析的實驗結果,EBNA2會增加CD300a啟動子活性。由於在細胞中EBNA2通常藉由跟轉錄因子RBP-Jκ互動,而達成調控EBNA2的標的基因表現,我們利用RNA干擾抑制RBP-Jκ在LCLs的表現,發現CD300a表現量有下降的情形。暗示了RBP-Jκ應該是參與在EBNA2所誘發的CD300a調控。 為了探討CD300a在LCLs的生理功能,我們在LCLs中利用RNA干擾抑制CD300a的表現。實驗結果顯示,抑制CD300a會使EB病毒進入溶裂期,暗示CD300a具有抑制EB病毒再活化的功能。且在螢光酵素報導分析中,實驗結果顯示,CD300a可以抑制Zta promoter (Zp)活性。因此我們認為CD300a可透過抑制Zp阻止EB病毒的再活化。另外本研究也發現利用RNA干擾的方式抑制CD300a後,會誘使細胞凋亡(apoptosis)及增加細胞激素IL-6 (Interleukin 6)的表現。而為了探討CD300a是透過何種路徑調控其下游訊號,根據RNA干擾的實驗結果,我們發現抑制CD300a同時也會造成JNK (c-Jun N-terminal kinase)、ATF2 (cAMP-dependent transcription factor)及Akt的磷酸化程度上升,並且抑制JNK磷酸化會降低抑制CD300a引起的EB病毒溶裂期蛋白表現,暗示CD300a可透過抑制JNK幫助EB病毒維持在潛伏期。而其餘磷酸化程度的變化對於EB病毒再活化及LCLs的生存的生理意義則有待討論。 As a human oncogenic virus, Epstein-Barr virus (EBV) infection is associated with many human diseases, especially in B cells disorders, such as Burkitt’s lymphoma, Hodgkin’s disease, post-transplant lymphoproliferative disorder (PTLD), diffuse large B cell lymphoma (DLBCL) etc. In vitro, the oncogenecity of EBV can be evidenced by its ability of immortalizing primary B cells into unlimitedly proliferating lymphoblastoid cell lines (LCLs). There are so many similarities between LCL and PTLD. So, LCLs become the perfect in vitro model to study EBV carcinogenesis. In our previous study, cDNA microarray is used to analyze the gene expression alteration between CD19+ primary B cells and LCLs. As expected, an array of genes display differentially between primary B cells and LCLs. Among them, a surface cluster of differentiation marker CD300a, mRNA expression is increased significantly in LCLs. Of interest, the up-regulation of CD300a only shows in B cells infected with EBV but not with other B cell stimulators, such as anti-CD40/ IL4, poly I:C, or LPS. So, we focus in the role of CD300a since it functions as an inhibitory receptor and involves in many regulation of immune activity in immune cells. In previous study, EBV nuclear antigen 2 (EBNA2) induces CD300a expression by transfection among the test viral genes, at transcriptional level. Data of luciferase reporter assay demonstrates that EBNA2 may up-regulate CD300a promoter activity. Usually, EBNA2 interacts with a cellular transcriptional factor RBP-Jκ to regulate gene expression. We find that RBP-Jκ is involved in EBNA2 mediated CD300a upregulation since knockdown of RBP-Jκ in LCL results in decrease of CD300a expression. In order to investigate the function of CD300a in LCLs, we knockdown CD300a by shRNA approach. We demonstrate that EBV enters the lytic cycle after knockdown of CD300a. It hints that CD300a inhibits EBV reactivation in LCL. Further, we show that CD300a can inhibit Zp (promoter of Zta) activity by luciferase reporter assay. We assume that CD300a inhibits EBV reactivation by repressing Zp activity. In addition, we find that cleavage of apoptotic protein PARP and IL-6 (Interleukin 6) production are increased when CD300a is knockdown. Mechanistically, we try to reveal which signaling pathways are triggered in CD300a downstream. According to results of CD300a knockdown, the phosphorylation level of JNK (c-Jun N-terminal kinase), ATF2 (cAMP-dependent transcription factor) and Akt is increased. The other data shows that inhibition of JNK activity restores the ability of CD300a to inhibit EBV reactivation. The biological meaning of this phosphorylation in EBV reactivation and LCL survival will be discussed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79056 |
DOI: | 10.6342/NTU201802958 |
全文授權: | 未授權 |
電子全文公開日期: | 2023-10-09 |
顯示於系所單位: | 微生物學科所 |
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