臺灣博碩士論文加值系統

葡聚醣 (β-glucan) 為多個葡萄糖組成的聚合物，廣泛地存在於植物、藻類、細菌及真菌類裡。過去有研究指出，β-glucan除了能降低發炎反應之外，也具有降低人體血清中低密度脂蛋白膽固醇含量，增加腸胃道功能和免疫調節作用。但β-glucan會因為來源屬性的不同，使得結構、分子量大小會有差異，進而影響每種β-glucan的功能性。儘管，目前為止有許多研究方向探討各種β-glucan的功能性，但較少有文獻比較不同來源之β-glucan抑制發炎反應之能力。因此，本研究依來源和結構的不同，以穀類(大麥)、藻類(昆布)、真菌類(酵母菌)三種β-glucan為研究樣本，比較其三種由LPS誘發巨噬細胞Raw 264.7 為發炎細胞模式其發炎因子之效果。本實驗方法以反轉錄聚合酶連鎖(Reverse transcription-PCR, RT-PCR)反應及酵素免疫分析法 (Enzyme-linked immunosorbent assay, ELISA)分析細胞中之發炎因子如：ICAM-1、MCP-1、IL-1β、NF-κB及iNOS之表現量。
　　在LPS誘發Raw 264.7發炎反應之細胞模式下，發現大麥β-glucan濃度150、200μg/mL時，能顯著減少IL-1β、NF-κB的表現量。昆布β-glucan濃度100μg/mL時，與LPS誘發組相比，能顯著抑制ICAM-1的表現量; IL-1β的表現量，當昆布β-glucan濃度150、200μg/mL時，能被顯著抑制。
酵母菌β-glucan的部分，除了NF-κB之外，發炎因子ICAM-1、MCP-1、IL-1β的表現量，在酵母菌β-glucan濃度200μg/mL時會被顯著抑制。
　　接著依濃度不同，來比較三種β-glucan抑制發炎因子的效果，發現
酵母菌β-glucan於濃度200 μg/mL相較於大麥和昆布β-glucan，前者較能顯著抑制ICAM-1、MCP-1、IL-1β這三種發炎因子。在NF-κB的部分，三種β-glucan於濃度50、100、150 μg/mL時，NFκB表現量下降之幅度沒有顯著差異。
　　由以上結果推測本實驗之結論，三種不同來源穀類(大麥)、藻類(昆布)、真菌類(酵母菌) β-glucan中，酵母菌β-glucan相較於大麥及昆布β-glucan，酵母菌β-glucan最能抑制發炎因子ICAM-1、MCP-1、IL-1β的表現量，同時也證實β-glucan確實能抑制發炎反應之相關因子。

β-glucans are polymers of glucose linked by glycosidic bonds, they are found in plants, bacteria and fungal. It might affect their property due to different sources and structures. Therefore, the objective of this study was to investigate three different kinds of β-glucans (barley, seaweed, yeast-derived β-glucan) and compare their anti-inflammatory effect to LPS-induced inflammation in Raw 264.7 cell. The inflammatory factors analyzed by the application of reverse transcription-PCR and enzyme-linked immunosorbent assay in this study included intercellular adhesion molecule 1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), nuclear factor-kB (NF-κB) and inducible nitric oxide synthase (iNOS).
The results showed that barley-derived β-glucan at 150, 200 μg/mL decreased production of IL-1β and NF-κB. Seaweed-derived β-glucan suppressed the content of ICAM-1 at 100 μg/mL and inhibited the content of IL-1β at 150, 200 μg/mL. However, yeast-derived β-glucan reduced the content of ICAM-1, MCP-1, IL-1β at concentration of 200 μg/mL.
In addition, yeast-derived β-glucan compared with barley-derived β-glucan, seaweed-derived β-glucan at 200 μg/mL showed the better inhibitory effect on ICAM-1, MCP-1, IL-1β. However, there were no significant differences on NF-κB at concentration of 50, 100, 150 μg/mL of three different kinds of β-glucans.
In conclusion, the comparative results of β-glucans in this study indicated that yeast-derived β-glucan has better inhibitory effects on production of ICMA-1, MCP-1, IL-1β under LPS-induced inflammation in Raw 264.7 cell.

目錄
中文摘要.........I
Abstract......III
目錄............IV
表目錄..........XII
圖目錄.........XIII
縮寫總表.........XV
壹、前言..........1
1.1研究動機.......1
1.2 研究目的......3
貳、文獻回顧.......4
2.1 葡聚醣簡介.....4
2.1.1 穀類β-glucan ( cereal β-glucan )...4
2.1.2 真菌β-glucan (fungal β-glucan).....6
2.1.3 藻類β-glucan (seaweed β-glucan)....7
2.2 發炎反應.......9
2.2.1 發炎反應簡介 ..9
2.2.2 脂多醣與巨噬細胞....10
2.2.3 發炎反應相關因子.....11
參、實驗材料與方法..17
3.1 實驗架構......17
3.2 實驗材料......18
3.2.1材料........18
3.2.2 化學試劑....18
3.2.3 儀器及相關設備.......19
3.3 實驗方法......20
3.3.1 細胞試驗....20
3.3.2 MTT細胞存活率試驗....21
3.3.3 一氧化氮含量分析......22
3.3.4 介白素-1β (Interleukin 1β, IL-1β)含量分析....22
3.3.5 細胞黏附分子(Intercellular Adhesion Molecule 1, ICAM-1)含量分析...23
3.3.6 單核球趨化蛋白(Monocyte Chemotactic Protein-1, MCP-1)含量分析......23
3.3.7 核因子-κB (Nuclear Factor-Kappa B, NF-κB)含量分析....24
3.3.8 Reverse transcription polymerase chain reaction....25
3.4 統計分析......28
肆、結果...29
4.1. β-glucan對Raw 264.7 細胞存活率之影響....29
4.2 大麥β-glucan對發炎因子含量的影響..30
4.2.1 大麥β-glucan對LPS誘導Raw 264.7 巨噬細胞ICAM-1含量之影響.. 30
4.2.2大麥β-glucan對LPS誘導Raw 264.7 巨噬細胞MCP-1含量之影響...30
4.2.3大麥β-glucan對LPS誘導Raw 264.7 巨噬細胞IL-1β含量之影響...30
4.2.4大麥β-glucan對LPS誘導Raw 264.7 巨噬細胞NF-κB含量之影響...31
4.3 昆布β-glucan對發炎因子含量的影響..31
4.3.1 昆布β-glucan對LPS誘導Raw 264.7 巨噬細胞ICAM-1含量之影響.. 31
4.3.2 昆布β-glucan對LPS誘導Raw 264.7 巨噬細胞MCP-1含量之影響..32
4.3.3 昆布β-glucan對LPS誘導Raw 264.7 巨噬細胞IL-1β含量之影響..32
4.3.4 昆布β-glucan對LPS誘導Raw 264.7 巨噬細胞NF-κB含量之影響..32
4.4 酵母菌β-glucan對發炎因子含量的影響........33
4.4.1酵母菌β-glucan對LPS誘導Raw 264.7 巨噬細胞ICAM-1含量之影響.. 33
4.4.2酵母菌β-glucan對LPS誘導Raw 264.7 巨噬細胞MCP-1含量之影響..33
4.4.3酵母菌β-glucan對LPS誘導Raw 264.7 巨噬細胞IL-1β含量之影響..34
4.4.4酵母菌β-glucan對LPS誘導Raw 264.7 巨噬細胞NF-κB含量之影響..34
4.5 比較各β-glucan於不同劑量對LPS誘導Raw 264.7巨噬細胞ICAM-1含量之變化....35
4.5.1 β-glucan於50μg/mL對LPS誘導Raw 264.7巨噬細胞ICAM-1含量之影響......35
4.5.2 β-glucan於100 μg/mL對LPS誘導Raw 264.7巨噬細胞ICAM-1含量之影響.....35
4.5.3 β-glucan於150 μg/mL對LPS誘導Raw 264.7巨噬細胞ICAM-1含量之影響.....35
4.5.4 β-glucan於200 μg/mL對LPS誘導Raw 264.7巨噬細胞ICAM-1含量之影響.....36
4.6. 比較各β-glucan於不同劑量對LPS誘導Raw 264.7巨噬細胞MCP-1含量之變化.....36
4.6.1 β-glucan於50 μg/mL對LPS誘導Raw 264.7巨噬細胞ICAM-1含量之影響......36
4.6.2 β-glucan於100 μg/mL對LPS誘導Raw 264.7巨噬細胞MCP-1含量之影響......37
4.6.3 β-glucan於150 μg/mL對LPS誘導Raw 264.7巨噬細胞MCP-1含量之影響......37
4.6.4 β-glucan於200 μg/mL對LPS誘導Raw 264.7巨噬細胞MCP-1含量之影響......37
4.7. 比較各β-glucan於不同劑量對LPS誘導Raw 264.7巨噬細胞IL-1β含量之變化.....38
4.7.1 β-glucan於50 μg/mL對LPS誘導Raw 264.7巨噬細胞IL-1β含量之影響.......38
4.7.2 β-glucan於100 μg/mL對LPS誘導Raw 264.7巨噬細胞IL-1β含量之影響.......38
4.7.3 β-glucan於150 μg/mL對LPS誘導Raw 264.7巨噬細胞IL-1β含量之影響.......39
4.7.4 β-glucan於200 μg/mL對LPS誘導Raw 264.7巨噬細胞IL-1β含量之影響.......39
4.8比較各β-glucan於不同劑量對LPS誘導Raw 264.7巨噬細胞NF-κB含量之變化.......39
4.8.1 β-glucan於50 μg/mL對LPS誘導Raw 264.7巨噬細胞NF-κB含量之影響.......39
4.8.2 β-glucan於100 μg/mL對LPS誘導Raw 264.7巨噬細胞NF-κB含量之影響.......40
4.8.3 β-glucan於150 μg/mL對LPS誘導Raw 264.7巨噬細胞NF-κB含量之影響.......40
4.9大麥β-glucan對發炎因子mRNA表現量之影響.....41
4.9.1大麥β-glucan對LPS誘導Raw 264.7 巨噬細胞ICAM-1 mRNA之影響..41
4.9.2大麥β-glucan對LPS誘導Raw 264.7 巨噬細胞MCP-1 mRNA之影響.. 41
4.9.3大麥β-glucan對LPS誘導Raw 264.7 巨噬細胞IL-1β mRNA之影響.. 42
4.9.4大麥β-glucan對LPS誘導Raw 264.7 巨噬細胞iNOS mRNA之影響..42
4.10昆布β-glucan對發炎因子mRNA表現量之影響..42
4.10.1 昆布β-glucan對LPS誘導Raw 264.7 巨噬細胞ICAM-1 mRNA之影響..42
4.10.2昆布β-glucan對LPS誘導Raw 264.7 巨噬細胞MCP-1 mRNA之影響..43
4.10.3昆布β-glucan對LPS誘導Raw 264.7 巨噬細胞IL-1β mRNA之影響..43
4.10.4昆布β-glucan對LPS誘導Raw 264.7 巨噬細胞iNOS mRNA之影響..43
4.11酵母菌β-glucan對發炎因子mRNA表現量之影響..44
4.11.1酵母菌β-glucan對LPS誘導Raw 264.7 巨噬細胞ICAM-1 mRNA之影響..44
4.11.2酵母菌β-glucan對LPS誘導Raw 264.7 巨噬細胞MCP-1 mRNA之影響..44
4.11.3酵母菌β-glucan對LPS誘導Raw 264.7 巨噬細胞IL-1β mRNA之影響..45
4.11.4酵母菌β-glucan對LPS誘導Raw 264.7 巨噬細胞iNOS mRNA之影響..45
伍、討論..57
5.1 β-glucan於細胞存活率之影響.....57
5.2 β-glucan抗發炎特性之影響.......58
5.2.1 大麥β-glucan抗發炎特性之影響..58
5.2.2 昆布β-glucan抗發炎特性之影響..59
5.2.3 酵母菌β-glucan抗發炎特性之影響 ..60
5.3 依相同劑量比較各β-glucan抗發炎之影響.......61
5.3.1 比較β-glucan於相同劑量對ICAM-1含量之影響.......61
5.3.2 比較β-glucan於相同劑量對MCP-1含量之影響........61
5.3.3 比較β-glucan於相同劑量對IL-1β含量之影響........62
5.3.4比較β-glucan於相同劑量對NF-κB含量之影響..62
5.4 綜合討論......63
陸、結論..65
柒、參考文獻.......66

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