臺灣博碩士論文加值系統

慢性發炎反應被認為會透過發炎性細胞激素來促進腫瘤細胞的增生、存活與轉移。於EB病毒相關的鼻咽癌中，也發現到了類似發炎反應的現象以及發炎性細胞激素的產生，但EB病毒如何促成此現象仍未完全明瞭。EB病毒的潛伏期膜蛋白-1 (latent membrane protein 1, LMP1) 已被認為是一致癌蛋白，它會活化多種細胞訊息路徑，進而調控許多細胞基因的表現，因此我們希望知道LMP1是否能調控鼻咽癌細胞表現發炎相關的細胞激素。首先，以蛋白質微陣列做初步的檢驗，結果顯示LMP1能促進鼻咽癌細胞株分泌八種細胞激素。其中MIP-1 alpha、MIP-1 beta與GM-CSF為三個新發現會受到LMP1調控的細胞激素，故被選為深入研究的對象。我們發現外源性的LMP1可以誘發鼻咽癌細胞和何杰金氏淋巴癌細胞產生MIP-1 alpha、MIP-1 beta與GM-CSF，同時證明在EB病毒感染的鼻咽癌細胞中，內生性的LMP1也會促進這三種細胞激素的表現。分析LMP1在鼻咽癌細胞株中所能活化的訊息傳遞路徑，發現canonical nuclear factor-kappa B (NF-kappa B)、non-canonical NF-kappa B以及c-Jun N-terminal kinase (JNK) 訊息路徑能顯著且一致的被活化。LMP1位於細胞質內部的羧基端有兩段C-terminal activating regions (CTARs)－CTAR1與CTAR2－負責活化細胞的訊息傳遞，我們也觀察到LMP1的CTAR1對活化non-canonical NF-kappa B是重要的，而CTAR2對於活化canonical NF-kappa B和JNK路徑是重要的。CTAR1或CTAR2缺失的LMP1突變蛋白會嚴重喪失其促進分泌MIP-1 alpha、MIP-1 beta與GM-CSF的能力。使用NF-kappa B與JNK訊息路徑抑制劑的結果，顯示活化NF-kappa B與JNK訊息路徑對於LMP1誘發這三種細胞激素是必需的。根據之前的報導指出，MIP-1 alpha和MIP-1 beta能夠吸引多種免疫細胞，包括大量浸潤在鼻咽癌中的T淋巴細胞。另外MIP-1 beta與GM-CSF在近期被發現似乎具有免疫調節的功能。因此，我們的研究結果暗示LMP1可能經由促進這些發炎性的細胞激素來控制鼻咽癌組織中的局部免疫反應。

Chronic inflammation can promote tumor cell proliferation, survival, and metastasis through inflammatory cytokines. Inflammation-like microenvironment and production of inflammatory cytokines have also been observed in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), but the contribution of EBV infection to this phenomenon is not fully understood. EBV latent membrane protein 1 (LMP1) has been considered an oncoprotein and can regulate many cellular genes through triggering multiple cell signaling pathways, so we wondered whether LMP1 can regulate expression of inflammatory cytokines in NPC cells. First, we utilized a protein array to examine the cytokine expression profiles, and eight cytokines were found to be upregulated by LMP1. Among these cytokines, MIP-1 alpha, MIP-1 beta, and GM-CSF were novel target genes of LMP1, so they were chosen for further study. We found that exogenous LMP1 induced production of MIP-1 alpha, MIP-1 beta, and GM-CSF in NPC and Hodgkin’s lymphoma cells, and also demonstrated that endogenous LMP1 enhanced expression of the three cytokines in EBV-infected NPC cells. Analyzing the signaling pathways activated by LMP1 in NPC cells, we found that the canonical nuclear factor-kappa B (NF-kappa B), non-canonical NF-kappa B and c-Jun N-terminal kinase (JNK) signaling pathways can be activated significantly and consistently. Within the intracellular carboxyl terminus of LMP1, two C-terminal activating regions (CTARs), CTAR1 and CTAR2, are responsible for activation of signal transduction. We also observed that CTAR1 was important for non-canonical NF-kappa B pathway, and CTAR2 was important for canonical NF-kappa�羠 and JNK pathways. LMP1 mutants with deletion at CTAR1 or CTAR2 significantly lost their ability to induce MIP-1 alpha, MIP-1 beta, and GM-CSF. Using inhibitors of NF-kappa B or JNK signaling pathways, we found that activation of NF-kappa B and JNK pathways were required for LMP1-induced production of the three cytokines. It has been reported that MIP-1 alpha and MIP-1 beta can recruit various immune cells, including T lymphocytes, which are dominant in the infiltrates in NPC. MIP-1 beta and GM-CSF may also act as immuregulatory factors in some circumstances. Therefore, our data suggest that LMP1 may modulate local immune responses in NPC tissues through upregulation of the inflammatory cytokines.

中文摘要------------------------------------------------- i
Abstract ---------------------------------------------- ii
目錄 -------------------------------------------------- iii
圖目錄--------------------------------------------------- v
序論----------------------------------------------------- 1
一、發炎反應與癌症--------------------------------------- 1
二、鼻咽癌----------------------------------------------- 2
三、EB病毒----------------------------------------------- 4
四、EB病毒潛伏期膜蛋白 LMP1------------------------------ 6
五、研究動機--------------------------------------------- 9
材料與方法---------------------------------------------- 10
質體DNA(plasmid DNA)與小干擾RNA(small interfering RNA, siRNA)-------10
質體轉型(transformation)---------------------------------10
製備質體DNA ---------------------------------------------10
細胞株(cell lines)以及細胞培養(cell culture) ------------11
細胞轉染(transfection) ---------------------------------12
蛋白質與細胞培養上清液之收取 ----------------------------12
蛋白質電泳與西方點墨法(SDS PAGE and Western blotting)--- 13
發炎相關激素蛋白質微陣列(Inflammatory cytokine arrya)--- 13
酵素免疫分析(Enzyme-linked immunosorbent assay, ELISA)---14
抗體---------------------------------------------------- 14
溶液---------------------------------------------------- 15
結果 ----------------------------------------------------16
一、LMP1誘發鼻咽癌細胞株分泌發炎相關的細胞激素---------- 16
二、LMP1促進鼻咽癌及何杰金氏淋巴癌細胞株分泌MIP-1α、MIP-1β與GM-CSF------------------------------------------------ 16
三、內生性的LMP1在感染EB病毒的細胞中與MIP-1α、MIP-1β及GM-CSF之分泌呈正相關 -------------------------------------- 17
四、內生性LMP1在EB病毒感染的細胞中促進與MIP-1α、MIP-1β及GM-CSF之分泌------------------------------------------------18
五、LMP1會活化鼻咽癌細胞中的NF-κB以及JNK訊息傳遞路徑---- 18
六、LMP1的CTAR1與CTAR2區域分別中介活化NF-κB或JNK訊息傳遞路
徑------------------------------------------------------ 19
七、LMP1的CTAR1和CTAR2區域對於促進分泌MIP-1α、MIP-1β及GM-CSF是重要的 ---------------------------------------------20
八、LMP1促進MIP-1α、MIP-1β及GM-CSF之分泌需要NF-κB與JNK訊息
路徑的活化---------------------------------------------- 21
討論---------------------------------------------------- 23
參考文獻------------------------------------------------ 30

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