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研究生:王詩綾
研究生(外文):Shih-ling Wang
論文名稱:芝麻素對人類單核球細胞表現氣喘相關趨化激素之影響及其表觀基因調控機制
論文名稱(外文):Sesamin Suppresses Macrophage-derived Chemokine Expression in Human Monocytes via Epigenetic Regulation
指導教授:趙大衛趙大衛引用關係洪志興洪志興引用關係
指導教授(外文):David ChaoChih-hsing Hung
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:64
中文關鍵詞:氣喘單核球細胞芝麻素表觀基因巨噬細胞衍生趨化激素趨化激素
外文關鍵詞:asthmamonocytessesaminepigeneticsCCL22chemokine
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趨化激素(chemokines)在氣喘的發病機制中扮演著重要的角色。芝麻素(sesamin)是一種從芝麻種子中純化出來的植物雌激素,在一些研究中發現與抗發炎有關,但芝麻素對氣喘相關的趨化激素是否有影響仍屬未知,本研究澄清芝麻素對人類單核球細胞表現干擾素γ誘導蛋白10(interferon-γ-inducible protein-10,IP-10/CXCL10)、巨噬細胞衍生趨化激素(macrophage-derived chemokine,MDC/CCL22)、生長相關癌基因α(growth-related oncogene-α,GRO-α/ CXCL1)和腫瘤壞死因子α(tumor necrosis factor-α,TNF-α)的影響。首先以芝麻素對細胞進行前處理,之後再以細菌脂多醣(lipopolysaccharide,LPS)刺激,利用酵素免疫分析法(ELISA)測定IP-10、MDC、GRO-α、TNF-α的濃度。為確認其調控機制,進行細胞表面受體之拮抗劑及細胞內路徑抑制劑、西方墨點分析(Western blot)和染色質免疫沉澱(chromatin immunoprecipitation,ChIP)等研究。結果顯示芝麻素抑制了MDC的表現,但對IP-10,GRO-α和TNF-α的表現沒有影響。給予雌激素受體(estrogen receptor,ER)拮抗劑和過氧化物酶體增殖物激活受體(peroxisomal proliferators-activated receptor,PPAR)-α拮抗劑會使被抑制的MDC表現回復。芝麻素可抑制細胞絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)中p38和核因子κB(nuclear factor κB,NFκB)中p65的磷酸化,亦可抑制組蛋白H3/H4在MDC啟動子區域的乙烯化。芝麻素可藉由通過ER/ PPAR-α,MAPK-p38和NFκB-p65的路徑抑制MDC的表現和表觀基因調控而抑制氣喘的發病。
Certain chemokines play important roles in asthma. Sesamin has anti-inflammatory effects. Whether sesamin modulates asthma-related chemokines is unknown. We investigated the effects of sesamin on interferon-γ-inducible protein-10 (IP-10/CXCL10), macrophage-derived chemokine (MDC/CCL22), growth-related oncogene-α (GRO-α/CXCL1) and tumor necrosis factor (TNF)-α expression in human monocytes. Cells were pretreated with sesamin and then stimulated with lipopolysaccharide (LPS). IP-10, MDC, GRO-α and TNF-α were measured by ELISA. Mechanisms were investigated by receptor antagonists, pathway inhibitors, Western blotting and chromatin immunoprecipitation. Sesamin suppressed the expression of MDC but had no effects on IP-10, GRO-α and TNF-α expression. The suppressive effect was reversed by the estrogen receptor (ER) and peroxisomal proliferators-activated receptor (PPAR)-α antagonists. Sesamin suppressed phosphorylation of mitogen-activated protein kinase (MAPK)-p38 and nuclear factor κB (NFκB)-p65. Sesamin suppressed histone H3/H4 acetylation in MDC promoter region. Sesamin may suppress asthmatic inflammation by suppressing MDC expression via the ER/PPAR-α, the MAPK-p38 pathway and the NFκB-p65 pathway and the epigenetic regulation.
論文審定書……....…………………………………………….…...…...…i
致謝……………………………………..………..........…………...……..ii
中文摘要……………………………………………………….…....……iv
英文摘要……………………………………..……………………......…..v
第 一 章 前言…………………………………………………..……....1
1.1 簡介………………………………………………………......…..1
1.2 單核球細胞(monocyte)…………………………………..…..2
1.3 芝麻素(sesamin)……………………………………..……….4
1.4 MAPKs及NFκB細胞訊息傳遞路徑……………….…………..6
1.5 組蛋白修飾(histone modification)……….…………………..7
第 二 章 研究目的………………………………..……………………9
第 三 章 材料與方法………………………………………..………..11
3.1 細胞培養...…………………………………………....................11
3.2 芝麻素的來源及配製…………………………….......................13
3.3 細胞存活率(Cell viability assay)……………........................14
3.4 酵素免疫分析法(Enzyme-linked immunosorbent assay,ELISA)…………………………………………………………..14
3.5 西方墨點分析法(Western blotting)……………………..….....15
3.6 染色質免疫沉澱法(Chromatin immunoprecipitation assay,ChIP).................................................................................................................17
3.7 聚合酶鏈鎖反應(polymerase chain reactio,PCR).....................18
3.8 數據統計分析................................................................................19
第 四 章 結果……………..…………………………………………..20
4.1 芝麻素會抑制LPS刺激後THP-1和human primary monocytes 表現MDC..................................................................................20
4.2 芝麻素對THP-1沒有細胞毒殺作用……………………..….21
4.3 芝麻素是經由ER和PPAR-α表面受體而抑制LPS誘導表現的MDC……………..…………………………………….…...22
4.4 MAPK抑制劑和NFκB抑制劑皆可抑制LPS誘導產生的MDC…………………………………………………………...22
4.5 芝麻素抑制LPS誘導人類單核球細胞表現的MDC是經由MAPK-p38和NFκB-p65訊息傳遞路徑………………...…...23
4.6 芝麻素經由抑制NFκB相關的乙醯轉移酶CBP抑制histone acetylation而使LPS誘導表現的MDC被抑制…………….24
第 五 章 討論………………………………………..……………..…26
第 六 章 結論……………………………………………..…………..32

參考文獻……………………………………………………...………….42
附錄………………………………………………………..……………..51
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