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研究生:楊乾隆
研究生(外文):Chien-Lung Young
論文名稱:重組真菌免疫調節蛋白與γ-次亞麻油酸對小鼠巨噬細胞之發炎介質生成的影響
論文名稱(外文):Effect of recombinant fungal immunomodulatroy protein-gts and gamma-linolenic acid on production of inflammatory mediators in Raw 264.7 macrophages
指導教授:黃永勝黃永勝引用關係
指導教授(外文):Yung-Sheng Huang
學位類別:碩士
校院名稱:元培科學技術學院
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:80
中文關鍵詞:重組真菌免疫調節蛋白r-次亞麻油酸DGLA花生四烯酸巨噬細胞
外文關鍵詞:fungal immunomodulatory proteinGamma-linolenic aciddihomo-GLAArachidonic Acidmacrophages
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發炎反應(inflammation)是宿主為了消滅外來物質和重建組織的一連串複雜與協同的反應過程,然而過度或慢性發炎往往會造成組織的損害及疾病的發生,例如過敏反應、自體免疫疾病。-次亞麻油酸 (gamma-linolenic acid, GLA) 被認為具有抗發炎以及調節免疫能力之功效。GLA在體內進行代謝形成dihomo-gamma linolenic acid (DGLA),接著在環氧化酶(cyclooxygenase)催化下形成前列腺素E1 (prostaglandin E1,PGE1),而具有抗發炎的作用。靈芝蛋白(LZ-8 protein)和真菌免疫調節蛋白(fungal immuno-modulatory protein-gts, FIP-gts)也被認為具有增強免疫能力之功能。然而靈芝菌株種類與萃取方法都會影響靈芝蛋白的品質,所以益生生技公司以基因工程方法開發並純化獲得重組真菌免疫調節蛋白(recombinant FIP-gts, rFIP-gts)。
目前對於rFIP-gts的研究還很少,所以本研究要探討rFIP-gts是否具抗發炎的作用?另外,由於人們可能同時攝取兩種以上的營養補充劑來調節其免疫機能,所以本研究也要探討rFIP-gts和GLA個別或併存時其抗發炎能力。本研究用細菌脂多醣(lipopolysaccharide, LPS)去刺激小鼠Raw 264.7巨噬細胞(macrophages)做為體外試驗模式,來觀察rFIP-gts與GLA對於LPS-stimulated macrophages之發炎介質(inflammatory mediators)生成量以及細胞脂肪酸組成的影響。
研究結果顯示:(1) rFIP-gts (10 ug/mL)對於LPS-stimulated macrophages的一氧化氮(NO)的產生沒有顯著的影響,而細胞激素IL-6、TNF-α等也則有上升的現象。GLA對於LPS-stimulated macrophages能抑制NO和細胞激素IL-6、TNF-α的生成。(3)當GLA與rFIP-gts和LPS-stimulated macrophages一起培養24小時會改變細胞的型態,且顯著的提高PGE1濃度而顯著的抑制PGE2濃度。(4)以固定100 M濃度的GLA和不同劑量的rFIP-gts一起處理LPS-unstimulated macrophages 24小時,並分析細胞脂肪酸的組成。結果顯示DGLA含量增多,同時arachidonic acid的含量下降,且此種現象與rFIP-gts的劑量呈現劑量反應的效應,故推測rFIP-gts可能會抑制Δ5-去飽和酶(Δ5-desaturase)活性。
綜合上述結果得知當GLA與rFIP-gts並存時,rFIP-gts主要是經由eicosanoids途徑而非藉由cytokines的途徑,來調節LPS-stimulated macrophages分泌發炎介質,故rFIP-gts具免疫調節功能。
Inflammation is a complex and coordinated response by the host to eliminate foreign materials and enact tissue repair. It encompasses aspects of both innate and adaptive immunity and has traditonally been described as a progression from acute to chronic stages followed by healing and reconstruction, such as allergy, autoimmune diseases. Gamma-linolenic acid (GLA) have efficiency of resisting the inflammation and regulating immune ability. GLA is elongated to form dihomo-GLA (DGLA), and the cyclooxygenase products of DGLA include prostaglandins of series 1 (PGE1) that have function of anti-inflammation. LZ-8 protein and fungal immunomodulatory protein-gts (FIP-gts) also has immune regulatory activities. However the kind of the ganoderma species and method of extracting will all influence the quality of ganoderma, Yeastern Biotech Company using the transgenic technology has successfully identified and isolated a specific yeast strain that can produce recombinant FIP-gts (rFIP-gts).

This study is rare of rFIP-gts, so this research confer the rFIP-gts wheather the function of anti- inflammation or not? In addition, people may use nutritious supplementary pharmaceutical of two kinds to regulate immune functions at the same time , The purpose of this study is also estimated the rFIP-gts and GLA in the regulation of LPS-induced inflammatory responses in RAW 264.7 macrophage, and observe rFIP-gts and GLA how to regulate inflammation mediators of LPS-stimulated macrophages and total cell fatty acid.

This results show: (1) rFIP-gts (100~ 0.3 ug/mL) no apparent influence the NO production of LPS-stimulated macrophages, but obvious effect of IL-6、TNF-α. (2)GLA can inhibit NO and IL-6、TNF-αcytokines of LPS-stimulated macrophages.(3)When GLA combine with rFIP-gts would change cell morphology,and raise the PGE1 and inhibit PGE2 level.(4)The concentration of GLA (100 mM) combine with different dosage of rFIP-gts to treat the LPS-unstimulated macrophages for 24 hours and analysis the compose of total cell fatty acid. It shows that increase DGLA but decrease Arachidonic Acid(ARA) yield. The effect is dosage dependent, and suggest that FIP-gts have beneficial effect against Δ5-desaturase.
Comprehensive these reasults show that GLA combine with rFIP-gts, rFIP-gts will regulate inflammation mediators of LPS-stimulated macrophages by eicosaoids but not cytokines, this indicate that rFIP-gts has immune regulatory activities.
目錄
中文摘要 I
Abstract II
目錄 III
圖目錄 IV
表目錄 V
第一章 緒論 1
1.1研究背景與研究動機 1
1.1.1發炎反應 1
1.1.2發炎與疾病 1
1.1.3發炎介質與發炎反應 3
1.1.4一氧化氮(Nitric oxide, NO)在發炎反應中扮演的角色 6
1.1.5花生四烯酸(Arachidonic acid, ARA)在發炎反應中扮演的角色 7
1.1.6靈芝(Ling Zhi)的研究背景 9
1.1.7 -次亞麻油酸 (gamma-linolenic acid, GLA) 16
1.2研究目的與重要性 18
第二章 研究方法 20
2.1實驗材料 20
2.2實驗藥品 20
2.3儀器設備 23
2.4實驗步驟 23
第三章 實驗結果 34
3.1 研究結果 34
3.1.1【實驗1】評估rFIP和GLA的劑量對un-stimulated macrophages之NO的生成量與細胞毒性的影響 34
3.1.2【實驗2】建立LPS-stimulated macrophages實驗模式 37
3.1.3【實驗3】 rFIP對LPS-stimulated RAW 264.7的影響 38
3.1.4【實驗4】評估rFIP與GLA並存下對於LPS-stimulated RAW 264.7 macrophage的影響 43
3.1.5【實驗5】rFIP和GLA濃度對RAW 264.7 macrophage 脂肪酸組成的影響 52
3.1.6【實驗6】rFIP和DGLA濃度對RAW 264.7 macrophage 脂肪酸組成的影響 59
第四章 討論 63
4.1體外評估rFIP對發炎物質的影響 63
4.2 FIP與GLA對於小鼠巨噬細胞脂肪酸代謝的影響作用 64
4.3 rFIP與 cell aggregation的影響 65
第五章 參考文獻 68
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