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研究生:葉子菱
研究生(外文):Tzu-Lin Yeh
論文名稱:輔助刺激因子與第四介白素在記憶型CD4+T淋巴球生成中所扮演的角色
論文名稱(外文):Role of co-stimulatory molecules and IL-4 in the generation of long-term CD4+ T cell memory
指導教授:孔祥智孔祥智引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:免疫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:53
中文關鍵詞:輔助刺激因子第四介白素記憶型 CD4+ T 淋巴球
外文關鍵詞:co-stimulatory moleculesIL-4memory CD4 T cell
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記憶型T淋巴球可抵抗對外來抗原,在免疫系統當中扮演重要的角色。 然而,對於控制記憶型CD4+ T淋巴球產生的詳細機制卻還不甚清楚。 在本研究當中,我們嘗試著去研究輔助刺激因子(costimulatory molecule)是否參與在記憶型CD4+ T淋巴球的生成。 經由cDNA的轉染(transfection)製造出表現在細胞表面帶有CD24、CD40、CD70、CD86、CD153、OX40L、4-1BBL和IcosL的EL4腫瘤細胞株。 另外,為了使這些轉染細胞株(transfectant)可以當作抗原呈現細胞(antigen-presenting cell)以活化CD4+ T淋巴球,因此在每種轉染細胞株的細胞表面上再加以轉染FcgRI,使轉染細胞株可以結合上anti-CD3單株抗體,並將之呈現給CD4+ T淋巴球以刺激之。 在活體外實驗的結果發現,除了CD28:CD86,在其他輔助型因子的刺激之下,CD4+ T淋巴球的活化狀況都不是很好。 因此在利用轉染細胞株EL4活化CD4+ T淋巴球時,我們同時加入anti-CD3和anti-CD28的刺激。 CD4+ T淋巴球在帶有各種不同輔助型刺激因子轉染細胞株EL4的刺激之後,再打入宿主老鼠體內,我們發現細胞表面表現CD40的轉染細胞株,可提供CD40:CD40L的刺激,造成大量長期存活的CD4+ T淋巴球。 另外,我們還發現CD4+ T淋巴球在B淋巴球的刺激之下再外加第四介白素也可以幫助生成長期存活的CD4+ T淋巴球。 至於其他的輔助刺激因子,包括CD28:CD86、CD27:CD70、CD30:CD153、OX40:OX40L、Icos:IcosL和4-1BB:4-1BBL,則對於記憶型CD4 T 淋巴球的生成沒有正向的影響。以上的研究發現對於腫瘤的治療與疫苗的開發皆有所幫助。
Long-lived memory T cells play important roles in the defense against foreign invaders, although the mechanisms involved in memory T cell generation are not fully understood. In this study, I studied the effects costimulatory molecules play on memory CD4+ T cell memory generation. EL4 tumor cell lines that had through transfection been made to stably co-express FcgRI and one or combinations of CD24, CD40, CD70, CD86, CD153, OX40L, 4-1BBL, and IcosL costimulators were used as antigen-presenting cells to activate CD4+ T cells. Such transfectants delivered signal 1 through anti-CD3 mAbs presented by its FcgRI and signal 2 through the given costimulatory molecules it expresses. Except CD28:CD86, all other costimulatory molecules were ineffective at causing CD4+ T cell proliferation. To insure proper activation of CD4 T cells, anti-CD3 and anti-CD28 were both added and were presented via the FcgRI on the various costimulator-expressing EL4 transfectants. Activated CD4+ T cells were adoptively transferred into histocompatible, congenic hosts and their presence was monitored over time. CD4+ T cells activated by anti-CD3/CD28 in the context of CD40:CD154 developed long-term survival potential. In addition, CD4+ T cells activated by anti-CD3/CD28 in the context of B cells + IL-4 also developed long-term survival potential. All other costimulators, CD28:CD86, CD27:CD70, CD30:CD153, OX40:OX40L, Icos:IcosL, and 4-1BB:4-1BBL, did not have positive effects on long-term CD4 memory generation. These results may have implications in tumor therapy and vaccine development.
Table of Contents

Abstract………………………………………………………………. v
Abstract (Chinese)…………………………………………………... vi
CHAPTER I Introduction………………………………………… 1
CHAPTER II Experimental Setup………………………..….…... 8
2.1 Mice…………….………………………………….………… 9
2.2 Isolation of spleen CD4+ T cells………………………….….. 10
2.3 RNA extraction and reverse transcription……………....……. 12
2.4 Cloning of CD24, CD40, CD153……………………………. 14
2.5 Transfection…………………………………………….……. 16
2.6 Preparation of Antigen-presenting cells (B cell blasts)…..….. 18
2.7 Activation of B10 CD4+ T cells………………………...……. 20
2.8 Adoptive transfer……………………………………….……. 21
2.9 Donor cell persistence traced by flow cytometry………..…... 22
CHAPTER III Experiment Results………………….…………... 23
3.1 Generation of EL4 lines that co-express FcgRI and different costimulatory molecules……………………………….…... 24
3.2 Using EL4 transfectants as APC to induce CD4+ T cells proliferation…………………………………………..……. 25
3.3 The effect of 4-1BBL, IcosL, CD70, and OX40L on the generation of memory CD4 T cells…………………….….. 26
3.4 The effect of IL-2 on the generation of memory CD4+ T cells ………………………………………………..…….. 27
3.5 The effect of IL-4 on the generation of long-lived T cells…… 28
3.6 The effect of combined costimulation signaling on the generation of memory CD4 T cells………………………… 29
3.7 The effect of CD40, CD153, OX40L on the generation of memory CD4+ T cells……………………………….……... 30
3.8 Rapid decline of donor T cells might be caused by rejecrion …………………………………………………. 30
3.9 Summary……………………………………………………... 31
CHAPTER IV Discussion………………….………………..……. 32
CHAPTER V Experiment Figures……………………………….. 39
REFERENCES……………………………………………………… 49
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