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研究生:楊雅倩
研究生(外文):Yang Ya-Chien
論文名稱:第一型人類嗜T細胞病毒(HTLV-I)臺灣株的分離與鑑定及HTLV-I感染之T細胞株對腫瘤壞死因子-a誘發細胞凋亡之敏感度的研究
論文名稱(外文):Isolation and Characterization of Taiwan Strains of Human T-lymphotropic Virus Type I (HTLV-I) and the Susceptibility of HTLV-I-infected T Cell Lines to Tumor Necrosis Factor-a-induced Apoptosis
指導教授:楊照雄楊照雄引用關係林榮華林榮華引用關係
指導教授(外文):Yang Czau-SiungLin Rong-Hwa
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:171
中文關鍵詞:第一型人類嗜T細胞病毒臺灣株腫瘤壞死因子-a細胞凋亡
外文關鍵詞:HTLV-ITaiwan strainstumor necrosis factor-aapoptosis
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  第一型人類嗜T細胞病毒 (human T-lymphotropic virus type I, HTLV-Ⅰ)是引起成人型T細胞白血病 (adult T cell leukemia, ATL) 及 HTLV-Ⅰ相關之脊髓病變/熱帶痙攣性下身輕癱 (HTLV-I-associated myelopathy or tropic spastic paraparesis, HAM/TSP) 的致病因。臺灣地緣上近於 HTLV-Ⅰ盛行地日本西南部,本地區 HTLV-Ⅰ感染的盛行率約為 0.48%,雖不是此病毒的流行區域,但各大醫院均可發現 ATL及 HAM/TSP病患;然而,臺灣一直未有分離出此病毒的報告。本論文先後自臺灣 HAM/TSP及 ATL病患的周邊血液單核細胞建立 HTLV-I陽性T細胞株,分別稱之為 THAM-1及 TATL-1,並對此二細胞株的表現型、細胞生長速度及細胞激素表現情形等特性進行研究,亦分析原病毒基因體嵌入細胞 DNA的情形及病毒 RNA和蛋白質的表現,也在電子顯微鏡下觀察到典型的C型反轉錄病毒顆粒;此外,亦完成了此二臺灣分離株 HTLV-ITHAM-1及 HTLV-ITATL-1之全部核酸序列的分析。
  HTLV-Ⅰ依據原病毒 LTR核酸序列分為美拉尼西亞型、薩伊型及全球型;全球型又可分為A亞型、B亞型和C亞型。為了進行臺灣地區 HTLV-I分子流行病學調查,本論文利用病毒外套基因第 5708至 6320核酸序列中兩個B亞型及四個C亞型特異性核酸,以巢式聚合鏈鎖反應及限制多形切點分析,建立一個簡單、快速的 HTLV-I亞型分子分型方法,並對四十一個臺灣抗體陽性檢體進行病毒分型;同時,上述檢體之聚合鏈鎖反應產物亦進行核酸序列分析,確定其六個亞型特異性核酸位置之核酸,結果顯示:所建立之分子分型方法完全符合核酸序列之分型。從研究中亦發現:臺灣地區存在有 HTLV-I全球A亞型及B亞型,而其中A亞型佔約 71%。
活化後或不正常的T細胞會藉由細胞凋亡作用而清除,已知有兩個途徑參與T細胞之細胞凋亡:一是 Fas及 Fas配體途徑;另一是腫瘤壞死因子-a (tumor necrosis factor-a, TNF-a) 及其受體 (tumor necrosis factor receptor, TNFR) 途徑。文獻已有報告:表現 HTLV-Ⅰ基因的T細胞株可降低對 Fas所引起細胞凋亡的敏感度。關於 TNF-a對 HTLV-Ⅰ感染之T細胞誘發細胞凋亡的情形則尚無報告。本論文先利用取自人類臍帶血的原生型T細胞進行 TNF-a誘發細胞凋亡的研究,新鮮分離出的原生型T細胞不表現 TNFR1及 TNFR2,經體外活化後此T細胞之細胞表面可測到微量的 TNFR1及較明顯的 TNFR2的表現;此活化後的T細胞可因 TNF-a的培養而發生細胞凋亡,而介白質-2可抑制此作用。TNF-a的兩種受體 TNFR1及 TNFR2皆可在人類活化T細胞中傳遞細胞凋亡的訊息,且二者具有加成作用。然而,不同於活化的T細胞,所研究的五種 HTLV-Ⅰ陽性T細胞株包括 HUT102、MJ、MT-2、THAM-1和 TATL-1皆無 TNFR1表現,並僅有 HUT102、MJ和 TATL-1可測得 TNFR2的表現;而此三種T細胞株對 TNF-a誘發之細胞凋亡作用皆具有抗性。此一避免細胞凋亡的機制可能與T細胞感染 HTLV-Ⅰ後仍可以存活進而有致病或癌化的機會有關。
Human T-lymphotropic virus type I (HTLV-I) has been implicated as the etiological agent of adult T cell leukemia/lymphoma (ATL) and certain neurological disorder named HTLV-I-associated myelopathy or tropic spastic paraparesis (HAM/TSP). Taiwan is geographically close to southwestern Japan, one of the endemic areas of HTLV-I infection. A sero-epidemiological survey in Taiwan showed that the prevalence of HTLV-I infection was 0.48%. Although Taiwan is a nonendemic region, patients with ATL or HAM/TSP have been sporadically encountered. However, the virus has not been isolated in Taiwan. In this dissertation, two HTLV-I-producing cell lines, designated THAM-1 and TATL-1, were established from the peripheral blood mononuclear cells of one HAM patient and one ATL patient, respectively. The characterization of THAM-1 and TATL-1, including phenotypes, growth rates and cytokine expression profiles, was studied. The analyses of proviral integration, viral RNA and HTLV-I-related antigens were also performed. Retrovirus particles with type C morphology were observed in these two cells. Meanwhile, the complete sequences of HTLV-ITHAM-1 and HTLV-ITATL-1 proviral genomes were determined.
According to the phylogenetic analysis of the long terminal repeat sequences of the virus, three types of HTLV-I have been proposed, (1) the Melanesian type, (2) the Zairian type, and (3) the so-called cosmopolitan type which has been further grouped into 3 subtypes: A, B and C. It is of interest to elucidate the molecular epidemiology of HTLV-I in Taiwan. In this dissertation, an easy and rapid method for identification of the cosmopolitan subtypes was developed by using a nested polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) studies using the 2 subtype B-specific and 4 subtype C-specific nucleotides located within the positions 5708 to 6320 of env gene. The nested PCR-RFLP method was used to subtype HTLV-I from 41 blood-unrelated Taiwanese. The sequences of PCR products were determined and the 6 positions of subtype-specific nucleotide variations were examined. The sequence data completely supported the subtyping data via the nested PCR-RFLP method. The results demonstrated that at least 2 distinct cosmopolitan subtypes (A and B) of HTLV-I were present in Taiwan and the more prevalent subtype in Taiwan is A (29 of 41; 70.7%).
Activated T cells during the downregulation of the immune response and T cells that become dysregulated may possibly be deleted by normal processes of elimination such as apoptosis. It is well documented that T cells may undergo apoptosis due to interactions between Fas and Fas ligand. Additionally, signals that induce apoptosis in T cells can result from tumor necrosis factor-a (TNF-a) and its receptors interaction. It has been shown that human T cell lines expressing HTLV-I have reduced susceptibility to anti-Fas-triggered apoptosis. The susceptibility of HTLV-I-infected cells to TNF-a-induced apoptosis remains to be investigated. In this dissertation, it was demonstrated that tumor necrosis factor receptors (TNFRs) were not detectable on naive T cells from cord blood. After in vitro activation, T cell blasts expressed low levels of TNFR1 and substantially higher levels of TNFR2 and these blasts were susceptible to TNF-a-induced apoptosis. The TNF-a-induced death of T cell blasts was potently inhibited by IL-2 added. Based on the agonistic effect of anti-TNFR1 and anti-TNFR2 antibodies, TNF-a-induced apoptosis involves an additive effect of both TNFR1 and TNFR2. Unlike activated T cells, TNFR1 was not detectable on all of the five HTLV-I-infected cell lines studied, including HUT102, MJ, MT-2, THAM-1 and TATL-1, and TNFR2 was only detectable on HUT102, MJ and TATL-1 cells. Nevertheless, HUT102, MJ and TATL-1 cells were resistant to TNF-a-triggered apoptosis. Protection from apoptosis may favor the survival and subsequent expansion of dysregulated HTLV-I-infected T cells with the potential to the development of HTLV-I-associated autoimmune-like inflammatory diseases or ATL.
封面
目錄
中文摘要
英文摘要
誌謝
縮寫/代號
表目錄(List of Tables)
圖目錄(List of Figures)
步驟目錄(List of Procedures)
第一章 導論
第一節 前言
第二節 第一型人類嗜T細胞病毒發現的歷史背景
第三節 第一型人類嗜T細胞病毒的基因體結構及其病毒蛋白質
第四節 第一型人類嗜T細胞病毒的生活史
第五節 第一型人類嗜T細胞病毒細胞嗜性及細胞受體
第六節 第一型人類嗜T細胞病毒的相關疾病
第七節 第一型人類嗜T細胞病毒的傳染途徑
第八節 第一型人類嗜T細胞病毒流行病學的研究
第九節 第一型人類嗜T細胞病毒的致病機轉
第十節 本論文主旨
第二章 第一型人類嗜T細胞病毒臺灣株的分離
第一節 前言
第二節 材料與方法
一.HAM/TSP病例
二.ATL病例
三.HTLV-I陽性細胞株之建立
四.其他國外建立之細胞株
五.免疫細胞化學染色法分析細胞表面抗原及HTLV-I病毒抗原
六.流式細胞儀分析細胞表面抗原及HTLV-I病毒抗原
七.電子顯微鏡觀察HTLV-I病毒顆粒
八.細胞DNA之抽取
九.細胞RNA之抽取
十.南方點墨雜交法分析HTLV-I原病毒基因
十一.北方點墨雜交法分析HTLV-I RNA
十二.放射活性探針之製備
十三.西方點墨分析法觀察HTLV-I抗原的表現
十四.MTT分析法測定細胞生長速度
十五.反轉錄-聚合?鏈鎖反應檢測細胞激素基因的表現
十六.寡核?酸五端放射活性標記法
第三節 結果
一.HTLV-I陽性細胞株之建立
二.HTLV-I病毒顆粒的觀察
三.HTLV-I嵌入原病毒基因體之分析
四.ATL癌細胞及TATL-1細胞TCRβ基因重新組合之比較
五.HTLV-I病毒RNA之分析
六.HTLV-I病毒抗原表現之分析
七.細胞生長速度之分析
八.細胞激素基因表現之分析
第四節 討論
第五節 本章總結
第三章 第一型人類嗜T細胞病毒臺灣株核?酸序列的分析
第一節 前言
第二節 材料與方法
一.HTLV-I臺灣分離株
二.國外已發表全長核?酸序列之HTLV-I分離株
三.利用PCR技術分段擴增HTLV-I原病毒基因體
四.HTLV-I原病毒基因體PCR產物的擇殖
五.HTLV-I核?酸序列之分析
六.系統發生分析
第三節 結果
一.HTLV-I臺灣分離株TATL-1及THAM-1原病毒基因體之完全核?酸序列
二.HTLV-I臺灣分離株TATL-1及THAM-1之核?酸序列與其他世界各地之分離株的親源性分析
第四節 討論
第五節 本章總結
第四章 以巢聚合?鏈鎖反應併限制?多形切點進行第一型人類嗜T細胞病毒亞型之分子分型
第一節
第二節 材料與方法
一.HTLV-I亞型巢式PCR/RFLP分子分型方法之設計
二.HTLV-I臺灣株檢體
三.PCR增幅產物之直接核?酸序列分析
四.HTLV-I系統發生分析
五.HTLV-I臺灣株env基因位置5708-6320之核?酸序列於基因庫的登錄號碼
第三節 結果
一.HTLV-I亞型巢式PCR/RFLP分子分型方法之建立及HTLV-I臺灣株亞型之分析
二.HTLV-I臺灣株部分env基因之直接核?酸序列分析
三.HTLV-I臺灣株與與其他世界各地分離株之系統發生分析
第四節 討論
第五節 本章總結
第五章 第一型人類嗜T細胞病毒感染之T細胞株對腫瘤壞死因子-α誘發細胞凋亡之敏感度的研究
第一節 前言
第二節 材料與方法
一.人類T細胞的來源
二.人類臍帶血T細胞的體外活化
三.研究的T細胞株
四.流式細胞儀分析細胞表面抗的表現
五.細胞凋亡的誘發
六.細胞凋亡的分析-TUNEL方法
七.細胞凋亡的分析-7-AAD染色方法
八.螢光顯微鏡觀察TNF-α受體與抗體結合後的聚集
第三節 結果
一.人類臍帶血及成人周邊血液T細胞CD45RA及CD45RO抗原的表現
二.人類TNF-αT細胞的體外活化後CD25及HLA-DR抗原的表現
三.人類臍帶血T細胞的體外活化前後TNFR1及TNFR2的表現
四.T細胞株之CD25及HLA-DR抗原的表現
五.T細胞株之TNFR1及TNFR2的表現
六.TNF-α誘發人類臍帶血之活化T細胞的細胞凋亡作用
七.TNFR1及TNFR2在TNF-α誘發人類臍帶血活化T細胞的細胞凋亡的角色
八.T細胞株對於TNF-α誘發細胞凋亡的敏感度
九.T細胞株之CD80(B7)抗原的表現
十.誘發細胞凋亡時的CD152Ig阻礙分析
十一.T細胞株之TNF-α受體與抗體結合後聚集的情形
第四節 討論
第五節 本章總結


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