臺灣博碩士論文加值系統

將抗原特異T細胞(Antigen-specific T cells)注射到病人體內來對抗一些癌症和病毒性疾病是一種相當具有潛力的治療方式，此種細胞治療方式不但可在試管內將選定的抗原特異細胞加以擴增，而避免在體內擴增的困難；同時亦可避免抗原特異T細胞在體內受腫瘤細胞或病毒所分泌的物質所抑制。
然而要能夠有效誘導及擴增抗原特異T細胞需要適當的抗原呈現細胞。抗原呈現細胞如樹狀細胞(Dendritic cell)就是一好刺激者，因為其呈現抗原能力高且具共刺激(co-stimulatory)及主組織相容性分子(major histocompatibility complex)。此外，相當多的研究發現，以樹狀細胞來活化受到抑制的T細胞時，樹狀細胞能恢復其活性。同時，過去的研究證實，患者體內的抗原特異T細胞的功能常不足夠，或是所展現功能的時期太短，以致沒有辦法將腫瘤或感染的細胞完全去除。因此本論文主要的目的是要以基因來改造抗原特異T細胞以增強其能力 。我們的作法是利用反轉錄病毒來將bcl-2基因送入抗原特異T細胞內，因為反轉錄病毒只會感染分裂中的細胞，而且bcl-2基因在細胞凋亡(Apoptosis)上扮演著相當重要的角色 。
本實驗利用B型肝炎病毒的表面(surface)及核(core)抗原基因，將樹狀細胞加以修飾，利用此修飾過的樹狀細胞去誘導並活化抗原特異T細胞。我們的結果發現具有B型肝炎抗原的樹狀細胞的確能專一地活化其抗原特異T細胞，而且反轉錄病毒亦能相對專一地將bcl-2基因送入B型肝炎特異T細胞，同時具有bcl-2基因的肝炎特異T細胞，其細胞存活率亦較高，亦能受到B型肝炎抗原的刺激而釋放干擾素g。

The infusion of antigen-specific T lymphocytes is a potential therapy against certain cancers and viral diseases. To increase their effectiveness, we examined whether the combined use of retroviral vector, which only infects dividing cells, and in vitro sensitization of T cells with antigen-loaded dendritic cells (DCs) could selectively modify antigen- specific T cells, and whether the transfer of bcl-2 gene could enhance the survival of antigen-specific T cells. The surface and core antigens of hepatitis B virus (HBV) were used as model antigens. DCs transfected with HBV antigens stimulated autologous T cell proliferation. Importantly, these proliferating autologous T cells could be selsctively transduced with bcl-2-retroviruses. After being subjected to apoptotic death by growth factor withdrawal, bcl-2-transduced T cells displayed enhance survival. These survival T cells were demonstrated to contain integrated bcl-2 provirus and exhibited antigen-specific interferon-g secretion. Therefore, the combined use of retroviral vector and T cell activation by antigen-loaded dendritic cells may selectively modify antigen-specific T cells and bcl-2 gene transfer into antigen-specific T cells may enhance their survival.

目錄
中文摘要
英文摘要
第一章、緒論 ------------------------------------------------------------------------ 1
第一節、研究動機及背景 ----------------------------------------------- 1
第二節、基因治療的研究發展 ----------------------------------------------- 3
1-2-1基因轉殖的方法
(一)病毒載體的方法
1.反轉錄病毒載體(Retroviral Vector)
2.腺病毒載體(Adenoviral Vector)
3.其他的病毒載體(Other Viral Vectors)
(二)非病毒載體的方法
1.微脂粒法之基因轉殖(liposome-mediated gene transfer)
2.受體引導之基因轉殖(Receptor-mediated gene transfer)
3.DNA直接注射至肌肉細胞
第三節、樹狀細胞(Dendritic cell, DC)-------------------------------------- 7
1-3-1樹狀細胞的特性
1-3-2樹狀細胞的來源
1-3-3樹狀細胞疫苗(Vaccine)的研發
第四節、細胞凋亡(Apoptosis)------------------------------------------------ 10
1-4-1細胞死亡的種類與特性
1-4-2細胞凋亡的基因調控
1-4-3判斷細胞凋亡的技術
第五節、預期完成的目標----------------------------------------------------- 13
第二章、材料與方法--------------------------------------------------------------- 14
第一節、細胞的製備與分化-------------------------------------------------- 14
2-1-1周圍血液單核球(Peripheral blood mono-nucleocytes, PBMC)的製備
2-1-2樹狀細胞(Dendritic cell)的分化
第二節、哺乳細胞表現載體(pcDNA3)的基因改造---------------------- 15
2-2-1 pcDNA3Δneo/HBVS的建構
2-2-2 pcDNA3Δneo/HBVC的建構
第三節、哺乳細胞表現載體的萃取----------------------------------------- 17
第四節、樹狀細胞的基因轉殖(Transfection)及基因表現分析--------- 19
2-4-1樹狀細胞的基因轉殖
2-4-2樹狀細胞的基因轉殖效率
2-4-3樹狀細胞的RNA的萃取
2-4-4樹狀細胞的RNA之RT-PCR分析
2-4-5樹狀細胞的cDNA的PCR分析
第五節、T細胞受活化增生之分析------------------------------------------- 23
2-5-1樹狀細胞之分化及基因轉殖
2-5-2 CD4+T淋巴球的製備
2-5-3 CD8+T淋巴球的製備
2-5-4單核球條件式培養液(Monocyte Conditioned Medium, MCM)
2-5-5 混和淋巴球反應(Mixed Leukocyte Reaction, MLR)
2-5-5a CD4+T細胞增生能力分析
2-5-5b CD8+T細胞增生能力分析
第六節、抗原特異T細胞(antigen-specific T cell)的基因轉導(transduction)--------------------------------------------------------- 28
2-6-1製備具有產生反轉錄病毒能力的細胞株
2-6-2 病毒數量(titer)分析
2-6-3 微脂粒基因轉殖法(SuperFect)
2-6-4 不含磷酸根(Phosphate-depletion)與微脂粒(LipofectAMINE)轉導方式的組合
2-6-5 CD25+T淋巴球的製備
2-6-6 T細胞的染色體DNA(genomic DNA)之萃取
第七節、基因轉導後T細胞染色體DNA之PCR分析---------------- 35
2-7-1 T細胞染色體的b-actin定量分析
2-7-2 T細胞染色體的neo基因的序列分析
第八節、Bcl-2基因表現分析------------------------------------------------ 37
2-8-1抗原特異T細胞的RNA萃取
2-8-2抗原特異T細胞的RNA之反轉錄作用(Reverse Transcription, RT)分析
2-8-3抗原特異T細胞的cDNA之PCR分析
2-8-4基因轉導後之抗原特異T細胞的活細胞計數
第九節、抗原特異T細胞之細胞間素(Cytokine)的測定及細胞增生之分析----------------------------------------------------- 40
2-9-1細胞間素的測定
2-9-2 活化(CD25+)之抗原特異T細胞的增生情形
第三章、結果------------------------------------------------------------------------ 43
第一節、樹狀細胞的基因轉殖效率與基因表現-------------------------- 43
3-1-1基因轉殖效率
3-1-2基因的表現
第二節、抗原特異T細胞的活化及bcl-2-neo基因轉導---------------- 44
3-2-1 T細胞的增生情形
3-2-2抗原特異T細胞的基因轉導
第三節、抗原特異T細胞的bcl-2基因表現、細胞間素分泌以及細胞增生情形-------------------------------------------------- 45
第四章、結論------------------------------------------------------------------------ 48
第一節、樹狀細胞的基因轉殖及基因表現
第二節、抗原特異T細胞的誘導
第三節、bcl-2-neo基因轉導及基因表現
第四節、抗原特異T細胞的IFN-g分析及增生情形
第五節、總結
第五章、參考文獻 ----------------------------------------------------------------- 52
表 ------------------------------------------------------------------------------------ 60
圖 ------------------------------------------------------------------------------------ 63
附圖一、本論文的實驗架構流程圖 ------------------------------------------- 88

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