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研究生:林雨涵
研究生(外文):Yu-Han Lin
論文名稱:葡聚醣、葡聚醣硫酸及葡聚醣硫酸鈉活化巨噬細胞探討
論文名稱(外文):Studies on the macrophages activation induced by Dextran, Dextran sulfate and Dextran sulfate sodium
指導教授:黃卓治黃卓治引用關係
指導教授(外文):Tzou-Chi Huang
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技系所
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:2013
畢業學年度:101
語文別:中文
論文頁數:66
中文關鍵詞:巨噬細胞葡聚醣葡聚醣硫酸葡聚醣硫酸去鈉葡聚醣硫酸鈉偽足電中性
外文關鍵詞:macrophage (RAW264.7)dextran (Dex)dextran sulfate (DS)desodium DSS (DSD)dextran sulfate sodium (DSS)synapseneutral and acids
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本研究的目的為鑑定葡聚醣化合物活化小鼠巨噬細胞(RAW 264.7)機制,利用文獻指出葡聚醣化合物可抗發炎、抗癌、誘導免疫活性增加,其方式為以免疫原具有類似脂多醣(Lipopolysacchadies, LPS)誘導巨噬細胞產生先天性免疫反應,但並不具有內毒素傷害細胞之特性,增加RAW 264.7之免疫調節活性,分別經由葡聚醣(Dextran, Dex)、葡聚醣硫酸(Dextran sulfate, DS)、去鈉葡聚醣硫酸(desodium, DSD)和葡聚醣硫酸鈉(Dextran sulfate sodium, DSS)對於免疫調節進行下列實驗之評估和比較,包括經由細胞毒殺作用(MTT assay)、一氧化氮表現量(Nitrite oxide assay)、抗原呈現分子主要組織相容性複合體(Major histocompatibility complex classⅡ)因子及另外兩種CD80和CD86因子、肌動蛋白細胞骨架的形態觀察(F-actin)、細胞的吞噬活性(Phagocytsis activity, FITC-E.coli.)、胞內自由基表現量(Reactive oxygen species, ROS),最後再觀察NF-κB轉移入核內之影響。結果顯示Dex, DS, DSD和DSS均對於小鼠巨噬細胞無傷害性,經由50-150 μM的濃度測定後其細胞存活率都可達95%以上,並且均可增加小鼠巨噬細胞的一氧化氮和胞內自由基產生量又為DSS最為明顯效果。此外,螢光顯微鏡觀察FITC-肌動蛋白細胞骨架的巨噬細胞吞噬Dex, DS, DSD和DSS的活性,在低濃度50-125 μM時Dex並無明顯偽足產生,但是在DS及DSS則在50-100 μM就有明顯偽足之產生。以流式細胞儀分析的方法來研究細胞表達CD80, CD86和MHC-Ⅱ類分子的螢光表現量,其結果透過統計數據顯示隨著加藥濃度提高則螢光表現均成正比,也由NF-κB轉移至核的結果顯示,此免疫原具有類似LPS的功能,能使60-75%的NF-κB轉移入核內,因此可以誘導產生iNOS使其產生Nitrite oxide則可誘導細胞先天性免疫反應產生,但不像LPS產生內毒素使細胞死亡。綜合來說,這些實驗證明Dex, DS, DSD和DSS可以對RAW 264.7有造成吞噬能力提高之表現,並藉由先天性免疫反應提高巨噬細胞的免疫調節功能和吞噬能力。研究顯示Dextran分子結構接上硫酸官能基後可增加其帶電性,因此對於誘導RAW 264.7免疫活性有顯著提升之表現,並證實葡聚醣化合物具有免疫調節之能力,未來將可致力於保健食品之開發與應用。
This study was designed to identify and characterize the mechanism of macrophage activation by dextran (Dex), dextran sulfate (DS), Desodium DSS (DSD) and dextran sulfate sodium (DSS). The immunomodulatory activity of Dex, DS, DSD and DSS, on mice macrophages were assessed and compared from various aspects including cytotoxicity, morphological of actin cytoskeleton, phagocytic activity, nitric oxide and major histocompatibility complex molecules in the absence of co-stimulation mediated via CD80 and CD86. Result showed that, Dex, DS, DSD and DSS were cytotoxin and could enhanced nitric oxide (NO) production in mice macrophages cells (RAW 264.7) in vitro. Furthermore, fluorescence microscope achieved the detection of actin cytoskeleton during macrophage phagocytosis when stimulation by Dex, DS, DSD and DSS. Detailed flow cytometric analysis was used to study the percentage of cell expressing CD80, CD86 and MHC-Ⅱ, were significantly great. Taken together, these experiments suggest that Dex, DS, DSD and DSS are required for confirmed the phagocytosis of macrophage. The microbial and cytokine milieu drives macrophages to express specialized and polarized functional properties. Lipopolysaccharides (LPS) elicit macrophage activation. This study was designed to identify and characterize the mechanism of macrophage activation by polysaccharides from dextran, dextran sulfate, desodium DSS, dextran sulfate sodium. The immunomodulatory activity of dextran polysaccharides on mice macrophages will be used to study the immunomodulatory activity of neutral, polysaccharide, Dex, DS, DSD, and DSS respectively. The immunomodulatory activity of polysaccharides were assessed and compared from various aspects including cell cytotoxicity test, morphological of actin cytoskeleton, NO expression. In the cell survival rate test, the survival rate is over 95% in 50-150 μM polysaccharides. By flow cytometry, major histocompatibility complex MHC-Ⅱ molecules, CD makers CD80, CD86 and the nitrite oxide ability increase with polyasccharides concentration. Final, synapse also can be found in fluorescence microscopy. All these experiments conducted aimed to elucidate the differential effect of neutral and acids 50-150μM Dex, DS, DSD and DSS on the activation of macrophage.
目 錄

摘 要......................................................I
Abstract.................................................III
誌 謝......................................................V
目 錄.....................................................VI
第壹章 前言................................................1
第貳章 文獻回顧............................................2
2.1 多醣類(Polysaccharide).............................. 2
2.1.1 葡聚醣化合物簡介................................... 3
2.1.2葡聚醣化合物與免疫反應相關性............................4
2.2免疫反應(Immune response)............................ 4
2.2.1先天性免疫反應(Innate immune response)................5
2.2.2適應性免疫反應(Adaptive immune response).............6
2.3巨噬細胞(Macrophage)..................................7
2.3.1吞噬作用(Phagocytosis)..............................8
2.4 MHC-Ⅱ與免疫相關性..................................9
2.4.1 CD80、CD86簡介...................................9
2.5脂多醣(Lipopolysaccharide, LPS).....................10
2.6 一氧化氮(Nitric oxide).............................11
2.6.1一氧化氮的形成及生理與病裡的特徵.....................12
2.7活性氧化物(Reactive oxygen species , ROS)............13
2.8 細胞骨架 (Cytoskeleton).............................15
第參章 研究動機..........................................16
第肆章 材料與方法..........................................17
4.1實驗架構..............................................17
4.2 細胞株 (Cell line)...................................18
4.3儀器設備..............................................18
4.4藥品試劑..............................................19
4.4.1 抗體..............................................19
4.4.2 樣品..............................................19
4.4.3 常用實驗藥品.......................................19
4.4.4 其他藥品............................................20
4.4.5 細胞實驗............................................20
4.4.6 細胞耗材............................................20
4.5實驗方法................................................21
4.5.1細胞解凍.............................................21
4.5.2細胞繼代培養.........................................21
4.5.3細胞計數..............................................21
4.6細胞存活率測定與分析(MTT assay)..........................21
4.7一氧化氮生成量測定與分析 (Nitric oxide assay)............23
4.8流式細胞儀測定MHC-Ⅱ的螢光表現量 ..........................25
4.8.1 流式細胞儀測定CD80的螢光表現量........................25
4.8.2流式細胞儀測定CD86的螢光表現量 ..........................26
4.9流式細胞儀測定葡聚醣化合物活化巨噬細胞吞噬能力..............27
4.10胞內ROS產量之分析.....................................27
4.11免疫螢光顯微鏡觀察偽足形成.............................28
4.12葡聚醣硫酸鈉去鈉鹽......................................30
第伍章 結 果..............................................31
5.1細胞存活率測定與分析(MTT assay).........................31
5.2一氧化氮生成量測定與分析(Nitric oxide assay)............31
5.3流式細胞儀測定MHC-Ⅱ的螢光表現量.........................32
5.3.1流式細胞儀測定CD80的螢光表現量.........................32
5.3.2流式細胞儀測定CD86的螢光表現量 .........................33
5.4流式細胞儀測定巨噬細胞吞噬之作用.........................34
5.5胞內ROS產量分析......................................35
5.6免疫染色螢光顯微鏡觀察偽足形成...........................36
5.7 NFκB核轉移之影響......................................37
第六章 討 論..............................................49
第七章 參考文獻...........................................52
作者簡介..............................................66

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