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研究生:賴宏瑋
研究生(外文):Hong-Wei Lai
論文名稱:香杉芝菌絲體多醣及其乳酸發酵液對Raw264.7與3T3-L1細胞交互作用之影響
論文名稱(外文):Effect of Antrodia salmonea mycelium polysaccharides and its lactic acid fermentation broth on the interaction between Raw264.7 and 3T3-L1
指導教授:毛正倫毛正倫引用關係
口試委員:蔡淑瑤梁志弘
口試日期:2019-06-13
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:102
中文關鍵詞:香杉芝菌絲體多醣胚芽乳酸桿菌Raw 264.73T3-L1共培養
外文關鍵詞:Antrodia salmonea myceliumpolysaccharideLactobacillus plantarumRaw 264.73T3-L1co-culture.
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肥胖伴隨的慢性發炎被認定為是導致許多慢性疾病之重要因子,包含高血壓、心血管疾病、胰島素阻抗、第2型糖尿病與部分癌症。慢性發炎的特徵為脂肪組織促發炎脂肪激素及趨化素分泌量上升,像是腫瘤壞死因子(TNF-α)、介白素-6 (IL-6)與單核球趨化蛋白-1(MCP-1);另一方面抗發炎激素生成量有所下降,如脂聯素。肥胖導致的發炎機制尚未完全了解,現階段研究大多指向細胞的壓力反應:舉凡缺氧、氧化壓力造成的傷害及脂肪細胞肥大導致的纖維化。肥胖的脂肪組織釋出高量的游離脂肪酸(FFA)、MCP-1及TNF-α等激素會在脂肪細胞與巨噬細胞的交互作用中形成惡性循環,進而導致胰島素阻抗、心血管疾病等慢性疾病。
香杉芝(Antrodia salmonea)為台灣特有種真菌,是於2004年被鑑定出的新品種,和藥用真菌牛樟芝(Antrodia cinnamomea)為同屬不同種的物種,多項研究顯示,牛樟芝萃取物具有多種生理活性功效,例如抗癌、抗氧化、抗發炎與抑制脂質分化等功能。鑑於香杉芝與牛樟芝整體性質特徵相似,因此可否利用培養成本較為低廉的香杉芝取代牛樟芝作為藥用資源就令人相當感興趣。

本次實驗主要探討香杉芝菌絲體多醣(PSAS)及其乳酸發酵液(USHKBC、CFS)相關生理活性功能,包含抗氧化能力分析、抗發炎與抗脂質分化影響。除此之外更進一步地探討巨噬細胞與脂肪細胞在共培養系統下相關發炎激素與發炎途徑相關蛋白表現量變化。
成分分析中,USHKBC及CFS的還原糖含量分別為3.89與3.92 % (w/w),而PSAS為0.79 % (w/w),結合發酵後乳酸菌數在添加PSAS的組別明顯高於未添加的組別,顯示香杉芝多醣能被Lactobacillus plantrumn所代謝。在DPPH自由基清除實驗中,USHKBC及CFS兩者之EC50分別為5.24%與5.15%,優於PSAS的13.48%。乳酸發酵液效果優於多醣樣品之結果也出現在還原力試驗中。
LPS誘導Raw 264.7發炎實驗中,一氧化氮(NO)生成量因樣品介入而下降,但USHKBC效果不如CFS及PSAS。細胞激素濃度方面,USHKBC、CFS、PSAS皆有抗發炎現象,不過劑量達到2.5%時抗發炎效果已大致達極限。在Raw 264.7與3T3-L1接觸式共培養模式中,給予脂肪酸誘導傷害後,USHKBC、CFS、PSAS皆未抑制NF-κB發炎途徑的蛋白表現,顯示抗發炎機制並非作用在此路徑上。
3T3-L1脂肪堆積實驗經油紅染色結果顯示USHKBC、CFS、PSAS皆會促進脂肪堆積,脂肪分化相關蛋白PPAR-γ2與C/EBPα表現量也有所上升。脂肪激素中,促發炎激素IL-6濃度上升而Leptin影響不大,抗發炎激素Adiponectin有所提升。在Raw 264.7與3T3-L1細胞間接式共培養系統中,介入USHKBC、CFS、PSAS作用24小時後,培養液中促發炎激素TNF-α、IL-6濃度遠高於控制組,而Adiponectin則有所提升。

綜合以上實驗結果,香杉芝菌絲體多醣及其發酵液具有抗氧化及抗發炎之功能,但抗發炎途徑並非作用在NF-κB發炎途徑上;另一方面,USHKBC、CFS、PSAS皆會促進脂肪堆積,因此在間接式共培養系統中導致促發炎激素濃度極高的原因推測是源自於樣品對3T3-L1的影響。
Obesity associated with chronic inflammation is recognized as an important risk factor for several chronic diseases, including hypertension, cardiovascular diseases, insulin resistance, type 2 diabetes and part of certain cancers. Chronic inflammation is characterized by the upregulation of proinflammatory adipokines and chemokines, such as tumor necrosis factor-α (TNF-α), interlukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in obese adipose tissuee. In contrast, adiponectin, an anti-inflammatory adipokine, is downregulated in obese adipose tissuee. The mechanism of chronic inflammation from obesity is currently fully unknown, but in previous studies point out that obesity-related inflammation are related to cellular stress responses, such as hypoxia, oxidative damages and hypertrophy-induced fibrosis. Free fatty acid (FFA), MCP-1 and TNF-α from obese adipose tissue establish a vicious cycle between adipocytes and macrophages, that further leads into chronic diseases like insulin resistance and cardiovascular diseases.
Antrodia salmonea is an endemic fungal in Taiwan. It’s a new species identified in 2004,which is the same genus with a medical fungal, Antrodia cinnamomea. The present studies have demonstraed that extracts of A. cinnamomea have lots of physiological function, such as anti-cancer, anti-oxidant, anti-inflammatory and anti-differentiation in adipocyte. In view of the characteristic are similar between A. salmonea and A. cinnamomea, therefore consideration of the cost-effectiveness, it’s interesting about whether A. salmonea could replace on A. cinnamomea.

The purpose of this study was to investigate the effects of A. salmonea mycelium polysaccharides (PSAS) and its lactic acid fermentation broth (USHKBC, CFS), fermentated by Lactobacillus plantarum on antioxidant activity, anti-inflammatory and anti-adipogenesis. Besides that, this study furthermore research about related inflammatory cytokines secretion and the expression of protein on inflamatory pathway in co-culture system.

The results showed that cotent of reducing sugar in USHKBC and CFS were 3.89 and 3.92 % (w/w) respectively, compared to 0.79 % in PSAS. Associated with numbers of lactic acid bacteria were greatly different between administration PSAS or without PSAS after fermentation. The results indicate that L. plantarum could metabolize PSAS. The IC50 of USHKBC and CFS in DPPH scavenging ability were 5.24 and 5.15% (v/v), which were better than 13.48 % in PSAS. The similar result were also found in reducing power experiment.
In the LPS-induced Raw 264.7 experiment, USHKBC, CFS and PSAS decreased Nitric oxide (NO) production, but USHKBC was weaker than the others. In the cytokines concentration test, USHKBC, CFS and PSAS existed anti-inflammatory properties, but the effective does was as limited as 2.5%. The expression of protein on NF-κB inflammatory pathway in contact coculture system between Raw 264.7 and 3T3-L1 didn’t be affected after administrating USHKBC, CFS and PSAS.

According to the result of oil red stain, USHKBC, CFS and PSAS could promote triglyceride accumutation, meanwhile C/EBPα and PPAR-γ2, related important proteins on adipocye differentiation, also expressed more strongly. Pro-inflammatory Adipokines, IL-6 was upregulatuon and Leptin wasn’t effected. Otherwise, adiponectin, an anti-inflammatory, was upregulatuon.

In conclusion, USHKBC, CFS and PSAS have anti-oxidant and anti-inflammatory effect. However, the mechanism of anti-inflammatory doesn’t work on NF-κB inflammatory pathway. On the other hand. USHKBC, CFS and PSAS could promote accumulation triglyceride, therefore the reason of pro-inflammatory cytokines were far greater than control group in indirect coculture system maybe attributed to the effects on 3T3-L1 cell lines.
摘要 i
Abstract ii
附圖及附表目錄 vi
表目錄 vii
圖目錄 viii
縮寫表 ix
第一章、 前言 1
第二章、文獻回顧 2
一、 香杉芝 2
(一) 香杉芝介紹 2
(二) 牛樟芝 2
(三) 香杉芝與牛樟芝之比較 2
二、 免疫反應 5
(一) 免疫系統 5
(二) 發炎 5
(三) 抗原辨識 7
(四) 細胞激素 9
(五) 氧化壓力與發炎 9
三、 脂肪細胞與組織 12
(一) 脂肪組織 12
(二) Adipokines 脂肪激素 12
(三) 脂肪細胞分化機制 15
(四) PPAR-γ 16
四、 肥胖與發炎 19
(一) 肥胖定義與評估 19
(二) 肥胖與慢性發炎 22
五、 菇類多醣 26
(一) 益生質 26
(二) 益生菌的免疫調節 26
(三) 菇類多醣結構與益生質 28
(四) 菇類多醣與肥胖相關慢性疾病 28
第三章、研究動機 30
第四章、實驗架構 31
第五章、材料與方法 32
一、 實驗材料 32
(一) 香杉芝菌絲體凍乾粉末 32
(二) 試藥 32
(三) 儀器 35
二、 實驗方法 36
(一) 樣品製備 36
(二) 除菌及破菌發酵液製備 36
(三) 成分分析 38
(四) 抗氧化實驗 38
(五) Raw 264.7細胞實驗 39
(六) 3T3-L1細胞實驗 41
(七) 西方墨點法 44
(八) 細胞共培養模式 44
三、 統計分析 45
第六章、結果與討論 46
一、 成分分析 46
二、 抗氧化分析 46
(一) 清除1,1-二苯基-2-苦味基團(DPPH)自由基能力 46
(二) 還原力試驗 50
(三) 螯合亞鐵離子能力 50
三、 Raw 264.7 巨噬細胞相關實驗 55
(一) 香杉芝菌絲體多醣及其乳酸發酵液對Raw 264.7 巨噬細胞存活率之影響 55
(二) 發炎調節性評估 55
(三) 促發炎/抗發炎細胞激素含量測定 61
四、 3T3-L1細胞實驗 66
(一) 香杉芝多醣及其發酵液對3T3-L1前脂肪細胞存活率之影響 66
(二) 油紅染色 (Oil red O stained material, OROSM) 66
(三) 脂肪激素(Adipokine)測定 69
(四) 脂肪分化蛋白(C/EBP α、PPAR-γ2) 72
五、 Raw 264.7 巨噬細胞與3T3-L1前脂肪細胞共培養相關實驗 77
(一) 細胞激素測定 77
(二) NF-κB發炎途徑蛋白表現 80
第七章、結論 84
第八章、參考文獻 85
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