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研究生:孟令敏
研究生(外文):Ling-Min Meng
論文名稱:樟芝多醣體對敗血症及肺部發炎反應免疫調節之影響
論文名稱(外文):Immunoregulatory effects of polysaccharides from Antrodia camphorata in sepsis and pulmonary inflammation
指導教授:葉松鈴葉松鈴引用關係
口試委員:許瑞芬蔡帛蓉蕭哲志劉珍芳
口試日期:2013-06-14
學位類別:博士
校院名稱:臺北醫學大學
系所名稱:保健營養學研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:150
中文關鍵詞:樟芝敗血症發炎細胞激素脾臟細胞激素 mRNA 表現多形核白血球、人類呼吸道上皮細胞株
外文關鍵詞:Antrodia camphorataPolymicrobial sepsisInflammatory cytokineSplenocyte cytokine mRNA expressionPMNsBEAS 2B cells
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樟芝為中國傳統中藥材,其所含多醣體曾被廣泛研究具調控免疫機制等各種生物功能,但目前並未有樟芝多醣體對於敗血症及其伴隨出現的肺臟損傷相關的研究。因此,本研究將實驗分為動物及細胞實驗兩部分,分別探討樟芝多醣體對敗血症生物體及肺上皮細胞發炎反應之影響。動物實驗以盲腸結紮及穿刺手術誘發小鼠產生敗血症,於術後 0.5 及 1 小時,依不同組別分別從腹腔注射生理食鹽水、100 mg/kg BW 樟芝菌絲或子實多醣體各二次,並於術後 6 及 16 小時,收取血液、腹腔沖洗液、肺臟及脾臟做分析。與一般控制組 (NC)相較,敗血症小鼠不論是血漿或腹腔沖洗液中 IL-10、IL-6、TNF-α 及 MCP-1皆明顯上升。手術 6 小時後,會造成小鼠白血球數量急遽下降;給予樟芝子實多醣體即可明顯提升血液中白血球數量,並降低腹腔沖洗液中 IL-6 濃度。於術後 16 小時,不論是菌絲或子實多醣體均可有效提升白血球數量、降低肺臟 NF-κB 蛋白表現量和腹腔沖洗液及血漿中 IL-10、IL-6、TNF-α 等 Th2 細胞激素的濃度;但僅有樟芝子實多醣體可顯著增加腹腔沖洗液中 IFN-γ 的濃度。而於脾臟細胞激素基因表現方面,術後 16 小時,與生理食鹽水組比較,不論菌絲或子實多醣體均可有效降低 TNF-α、IL-10 及 IL-6 的 mRNA 表現。由肺臟組織切片染色結果,發現菌絲或子實多醣體皆可減輕肺泡損傷,其中以子實體效果較好。
細胞實驗以脂多醣 (LPS) 刺激人類呼吸道上皮細胞 (BEAS 2B) 造成發炎反應來探討樟芝多醣體介入的影響。研究結果發現,樟芝多醣體可減少多型核白血球 (PMNs) 遷移至 BEAS 2B 的數目,並降低 PMNs 和 BEAS 2B 促發炎激素及 IL-8、MCP-1 的產生量。本結果顯示樟芝多醣體可能藉由抑制細胞分泌 IL-8,進而減少 PMNs 遷移至 BEAS 2B 細胞層,達到保護肺臟的作用。本研究結果顯示,樟芝多醣體可經由調節免疫反應以減緩呼吸道上皮細胞及敗血症之發炎反應,並減輕肺臟組織損傷。
Antrodia camphorata (AC) is a traditional Chinese medicine, and polysaccharides contained within AC (AC-PSs) are reported to possess various biological functions. At present, no study has evaluated the impact of AC-PSs on modulating the immune response in polymicrobial sepsis and associated lung injury. This study, through in vivo and in vitro experimental designs, investigated the immunoregulatory effects of AC-PSs extracts on sepsis and pulmonary inflammation. There were 1 normal control (NC) and 3 experimental groups. The NC group underwent a sham operation, whereas the experimental groups received cecal ligation and puncture (CLP) to induce sepsis. Mice in the experimental groups were further divided into saline, mycelia, and fruiting body treatment groups. Saline or AC-PSs were injected intraperitoneally twice at 0.5 and 1 h after CLP and were sacrificed at 6 or 16 h after sepsis for further analysis. Compared to the NC group, interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-10, and monocyte chemotactic protein (MCP)-1 levels in plasma and/or peritoneal lavage fluid (PLF) of septic mice dramatically increased after CLP. The elevated levels of these inflammatory mediators in both of the AC-PS-treated groups had decreased by 16 h after CLP. Messenger (m)RNA expressions of TNF-α, IL-6, and IL-10 in splenocytes were lower in both AC-PS-treated groups than in the saline group. Consistent with the results, lung nuclear factor-κB expressions decreased and less severe interstitial inflammation was observed in histological finding after CLP in mice that had received AC-PSs. Fruiting body group had higher white blood cell counts and lower IL-6 levels in PLF 6 h after CLP whereas interferon-γ (IFN-γ) was higher 16 h after CLP than in the saline group. These alterations were not found in mice injected with the mycelia extract. Administration of AC-PSs, either from mycelia or fruiting bodies, decreased inflammatory mediator expressions both at the location of injury and in the circulation, especially in the late stage of sepsis. AC-PSs from fruiting bodies seemed to be more effective in reducing inflammatory response than those from mycelia. The in vitro study investigated the effect of AC-PSs on lipopolysaccharide (LPS)-induced inflammatory response in human lung epithelial (BEAS 2B) cells and polymophonuclear cells (PMNs). The results showed that AC-PSs decreased LPS-induced BEAS 2B activation and recruitment of PMNs to BEAS 2B cells layer. Also, the expressions of inflammatory cytokine and IL-8, MCP-1 secreted by PMNs and BEAS 2B cells decreased. This study indicated that administration of AC-PSs attenuated the inflammatory reaction of BEAS 2B cells and PMNs that may consequently reduced the transepithelial migration of PMNs. These findings imply that AC-PSs from mycelia and fruiting bodies have potential protective effects against polymicrobial sepsis and lung inflammation.
第一章 前言 1
第一節 樟芝簡介 1
壹、分類法 1
貳、分佈及生長特性 3
參、毒性作用探討 4
肆、人工培育法 5
伍、生理活性成分及功效 7
第二節 人類之免疫防禦能力 9
第三節 敗血症簡介 12
壹、定義及分級 13
貳、引發敗血症相關機轉 15
參、敗血症肺臟損傷之探討 21
肆、敗血症之動物模式 29
第二章 研究動機與目的 31
第三章 不同來源樟芝多醣體成份分析研究 33
第一節 實驗目的 33
第二節 樟芝多醣體萃取法 33
壹、樣品取得 33
貳、實驗方法 34
第三節 樟芝子實及菌絲多醣體分析方法 34
壹、Phenol-sulfuric acid測定法 34
貳、HPLC-ELSD分析 35
參、LAL-endotoxin測定法 35
肆、結果 36
第四章 樟芝多醣體對敗血症小鼠之發炎反應、白血球數目及器官損傷之影響 38
第一節 實驗目的 38
第二節 實驗動物 38
壹、實驗動物品系選用依據 38
貳、飼養條件 39
參、CLP (cecal ligation and puncture) 手術 39
第三節 實驗方法 40
壹、存活率實驗 40
貳、肺臟組織切片實驗 40
参、樟芝對敗血症小鼠發炎反應實驗 42
第四節 統計方法 53
第五節 結果 53
壹、存活率實驗 53
貳、肺臟組織切片實驗 53
参、樟芝對敗血症小鼠發炎反應實驗 54
第六節 討論 65
第七節 結論 71
第五章 樟芝多醣體對呼吸道上皮細胞發炎反應之影響 72
第一節 實驗目的 72
第二節 樟芝濃度及時間測試實驗 73
壹、細胞株 73
貳、實驗設計 73
參、分析項目與方法 74
肆、統計方法 77
伍、結果與討論 77
第三節 樟芝多醣體對人類PMNs、BEAS 2B 細胞發炎反應之調控 84
壹、實驗與方法 84
貳、實驗設計 85
參、統計分析 87
肆、結果 88
伍、討論 94
陸、結論 97
第六章 總結 98
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