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研究生:林宛蓁
研究生(外文):Lin, Wan-chen
論文名稱:合成alpha-醣神經醯胺並研究其結構變異對活化自然殺手T細胞之影響
論文名稱(外文):Structural Variations of alpha-Glycosylceramides and Their Influence on Natural Killer T Cell Activation
指導教授:方俊民方俊民引用關係林俊宏林俊宏引用關係
指導教授(外文):Fang, Jim-minLin, Chun-hung
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:86
中文關鍵詞:alpha-醣神經醯胺自然殺手T細胞
外文關鍵詞:alpha-GlycosylceramidesNatural Killer T Cell
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CD1是一群在結構與功能上近似MHC(主要組織相容複合體)的醣蛋白,藉由呈獻疏水性的抗原分子(例如醣脂質)讓T細胞辨識而活化T細胞。研究顯示,人類的CD1d(CD1家族成員之一)以及其老鼠之同源體皆可呈獻一種特別的醣脂質,alpha-galactosylceramide(alpha-GalCer)給免疫系統中的自然殺手T細胞(natural killer T cell,簡稱NKT 細胞)。一般推測,抗原呈獻細胞(antigen presenting cells, 簡稱APC)表面的CD1d/alpha-GalCer複合體被NKT細胞表面之受體辨識,使NKT細胞被活化而釋放出大量的細胞激素包括IFN-gamma及IL-4,顯示NKT細胞很可能在免疫系統中扮演著調節性的角色。研究也顯示活化的NKT細胞可觸發免疫系統抵抗腫瘤之功能,對抑制自體免疫疾病也有著關鍵性的影響,使得NKT細胞之相關研究近年來受到眾多注目。
在過去關於NKT細胞活化的研究中,alpha-GalCer和alpha-GlcCer是活性最高的抗原分子。此類醣脂質分子數個影響活性之關鍵結構特徵已被指出,包括連結醣類與脂質的醣鍵位向(alpha-glycosidic linkage)、醣類二號位置之羥基與phytosphingosine上的兩個羥基都是alpha-GalCer具有活性的必要條件。抗原結構的改變可能導致抗原活性的增減,也可能影響NKT細胞活化後釋放的細胞激素種類。本論文之研究目標為alpha-GalCer及其結構類似物之合成與免疫測試,藉此更進一步探討alpha-GalCer之結構改變對其免疫活性之影響。在alpha-GalCer的製備方面,我們提出一個新的策略可同時應用於新一代類似物之合成。為了探討phytosphingosine上3’及4’位置羥基對活性的影響,我們也設計了數個類似物,將此部位之官能基以酯基或醯胺基替代,可輕易地由絲胺酸衍生物製備而成。此外,我們也探討了在醣類二號位置引進其他官能基的方法,提供往後合成新類似物的基礎。最後,我們利用酵素結合免疫吸附法(ELISA)測試類似物的免疫活性,並與alpha-GalCer的測試結果比較,以瞭解引入之結構變異對於化合物免疫活性之影響。

CD1 molecules are a family of MHC-like cell surface glycoproteins that present glycolipid antigens to T lymphocytes. Human CD1d and its mouse homolog are shown to present a special glycolipid, a-galactosyl ceramide (alpha-GalCer), to a subset of T lymphocytes called natural killer T (NKT) cells. It is suggested that NKT cells are activated through the TCR interaction with CD1d/ alpha-GalCer complex. NKT cells are thought to be immuno-regulators because they secrete large amounts of cytokines, such as IL-4 and INFgamma, after stimulation. They are also found to show antitumor activity and to control autoimmune responses.
Among the reported glycolipids presented by human CD1d (or its mouse homolog), alpha-GalCer and alpha-GlcCer show the highest immuno-stimulatory activities. Some structural features of alpha-GalCer have been demonstrated necessary for antigen recognition by CD1-restricted NKT cells, including: (1) the alpha- anomeric glycosidic linkage between sugar and lipid moieties; (2) the 2-hydroxyl group on the pyranose ring; and (3) the presence of 3’ and 4’-hydroxyl groups on the phytosphingosine chain. Change of the antigen structure may alter the level of stimulatory activity and/or switch the cytokine pattern released by NKT cells. However, the information is still limited about how antigen structures influence the activation event. In order to obtain the scenario about structure-activity relationship of alpha-GalCer, we synthesized alpha-GalCer and its analogues based on the above-mentioned features. The synthetic approach was designed not only to increase versatility of the alpha-GalCer synthesis but also to accommodate the flexibility for further modification. The phytosphingosine moiety was replaced by corresponding serine ester or amide as the simplified lipids to afford analogues ANGX1-3. In addition, the model study for functional group replacement at sugar C-2 position was demonstrated. Finally, an ELISA assay was established to evaluate the stimulatory activities of these analogues and compare the results with that of alpha-GalCer.

Acknowledgement ...............................................i
Abstract .....................................................ii
Abbreviations ................................................iv
Chapter 1. Introduction .......................................1
1.1 Biology of Natural Killer T Cells ........................1
1.2 Historic Review of Structural Investigations for alpha-
Glycosylceramides ........................................6
1.3 Immunological Methods for Evaluating the Activity of
alpha-Glycosylceramides .................................13
1.4 Design of Target Molecules ..............................16
Chapter 2. Results and Discussion ............................20
2.1 Synthesis of alpha-Glycosylceramides ....................20
2.1.1 Synthetic Strategies .................................20
2.1.2 Synthesis of Glycosyl Donors .........................24
2.1.3 Synthesis of Glycosyl Acceptors ......................25
2.1.4 Construction of the Glycolipid .......................29
2.2 Synthesis of Analogues .................................32
2.3 Model Studies for Synthesizing New Analogues with
Substituents on Sugar C2 Position .......................36
2.4 Bioassay ................................................39
2.4.1 Assay setup ..........................................39
2.4.2 Evaluation of Analogues ..............................40
2.5 Concluding Remarks ......................................42
Chapter 3. Experimental Section ..............................45
References ...................................................82
Curriculum Vitae .............................................86
Appendix: NMR Spectra of Products

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