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研究生:周孝芳
研究生(外文):Hsiao Fang Chou
論文名稱:漢黃芩素調控細胞因子誘發血清類澱粉蛋白A在人類肝癌細胞株HepG2中的表達-STAT3、NF-κB及C/EBPβ的角色
論文名稱(外文):Wogonin modulates cytokine-induced serum amyloids A protein expression in HepG2 cell lines-roles of STAT3, NF-κB and C/EBPβ
指導教授:張瑛玲張瑛玲引用關係
指導教授(外文):Y. L. Chang
學位類別:碩士
校院名稱:長庚大學
系所名稱:中醫學系傳統中醫學
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:104
語文別:中文
論文頁數:92
中文關鍵詞:血清類澱粉蛋白A漢黃芩素人類肝癌細胞株HepG2轉錄因子細胞因子
外文關鍵詞:Serum amyloid AWogoninHepG2 cell lineTranscription factorCytokines
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目錄
指導教授推薦書
論文口試委員會審定書
誌謝...............................................................................iii
中文摘要............................................................................iv
Abstract............................................................................v
中英文縮寫對照表(Abbreviations)....................................................................ix
第一章 緒論 (Introduction)............................................................1
1.1 類風濕性關節炎與類澱粉蛋白A沉積症 (RA and AA Amyloidosis)..........................1
1.2 發炎反應與血清類澱粉蛋白A (Inflammation and SAA)..................................3
1.3 血清類澱粉蛋白A的特性 (Characteristics of SAA)....................................4
1.3.1 信號轉導與轉錄活化蛋白-3 (STAT3)...............................................6
1.3.2 核因子-κB (NF-κB).............................................................6
1.3.3 CCAAT增強子結合蛋白β (C/EBPβ).................................................7
1.4 黃芩 (Huang-Qin).................................................................8
1.4.1 簡介.........................................................................8
1.4.2 成分.........................................................................9
1.4.3 現代藥理研究.................................................................10
1.5 漢黃芩素 (Wogonin)..............................................................11
第二章 研究目的 (Aim of the study)...................................................14
第三章 實驗假說 (Hypothesis).........................................................15
第四章 材料與方法 (Materials and Methods)............................................16
4.1 實驗材料......................................................................16
4.1.1 細胞株 (Cell line).........................................................16
4.1.2 實驗藥品 (Experimental drugs)..............................................16
4.1.3 實驗耗材 (Consumables).....................................................19
4.1.4 抗體試劑 (The Antibodies)..................................................19
4.1.5 儀器設備 (Equipment).......................................................20
4.2 實驗方法......................................................................22
4.2.1 藥品製備 (Preparation for drugs)...........................................22
4.2.2 細胞培養 (Cell Culture)....................................................22
4.2.3 蛋白質萃取 (Extraction of Protein)..........................................25
4.2.4 蛋白質定量 (Protein Assay)..................................................26
4.2.5 西方墨點法 (Western Blotting)...............................................27
4.2.6 免疫共沉澱法 (Co-Immunoprecipitation, Co-IP)................................30
4.2.7 RNA萃取與定量 (Extraction and Quantification of total RNA)..................31
4.2.8 反轉錄反應 (Reverse Transcription)..........................................31
4.2.9 定量即時聚合酶鏈鎖反應 (Quantitative real time PCR)..........................32
4.2.10 免疫螢光染色法 (Immunofluorescence staining)...............................33
4.2.11 統計分析 (Statistical Analysis)...........................................34
第五章 實驗結果 (Results)............................................................35
5.1 細胞因子IL-1β與IL-6單獨或併用對SAA mRNA表現量的影響..............................35
5.2 漢黃芩素對HepG2細胞受細胞因子刺激誘導之SAA mRNA及蛋白表現量的影響..................35
5.3 細胞因子活化轉錄因子的時間點變化.................................................36
5.4 漢黃芩素抑制細胞因子對NF-κB及C/EBPβ的活化........................................37
5.5 漢黃芩素抑制細胞因子誘導STAT3的磷酸化............................................37
5.6 漢黃芩素對血清類澱粉蛋白之轉錄複合物的影響........................................38
5.7 漢黃芩素抑制細胞因子對IκBα的磷酸化...............................................39
5.8 漢黃芩素對細胞素信號傳導抑制因子-3蛋白的影響......................................39
5.9 漢黃芩素不影響由IL1+6所活化之MAPK訊息傳導路徑.....................................40
第六章 討論 (Discussion).............................................................41
圖與附錄 (Figures and Appendixes)...................................................46
參考文獻 (References)...............................................................73

圖目錄
圖5.1 細胞因子IL-1β與IL-6單獨或併用對SAA mRNA表現量的影響..............................47
圖5.2 細胞因子IL-1+6刺激誘導之SAA mRNA及蛋白表現量的時間效應............................48
圖5.3 漢黃芩素對HepG2細胞受細胞因子刺激誘導之SAA mRNA及蛋白表現量的影響..................49
圖5.4 細胞因子活化轉錄因子的時間點變化.................................................50
圖5.5 漢黃芩素抑制細胞因子對NF-κB及C/EBPβ的活化........................................51
圖5.6 漢黃芩素對細胞因子誘發STAT3、NF-κB及C/EBPβ的影響.................................52
圖5.7 漢黃芩素抑制細胞因子誘導STAT3的磷酸化............................................53
圖5.8 漢黃芩素對血清類澱粉蛋白之轉錄複合物的影響........................................54
圖5.9 漢黃芩素抑制細胞因子對IκBα的磷酸化...............................................55
圖5.10 漢黃芩素對SOCS3蛋白的影響......................................................56
圖5.11 漢黃芩素不影響由IL1+6所活化之MAPK訊息傳導路徑....................................57
圖5.12 漢黃芩素Wogonin可能作用機制圖..................................................58
附錄1 類風濕性關節炎併發類澱粉沉積症之發病機制..........................................59
附錄2 急性期反應的活化...............................................................60
附錄3 人類A-SAA蛋白之結構............................................................61
附錄4 血清類澱粉蛋白A基因啟動子示意圖..................................................62
附錄5 JAK/STAT訊號傳遞路徑...........................................................63
附錄6 NF-κB訊號傳遞路徑與IκB降解作用..................................................64
附錄7 C/EBPβ訊號傳遞路徑.............................................................65
附錄8 黃芩藥材飲片圖.................................................................66
附錄9 漢黃芩素Wogonin之結構圖.........................................................67
附錄10 促發炎因子在類風濕性關節炎之致病角色.............................................68
附錄11 抗細胞激素療法用於治療類澱粉蛋白沉積症病程發展....................................69
附錄12 以細胞激素阻斷劑治療類風濕性關節炎之血清類澱粉蛋白A啟動子與血清中含量濃度示意圖......70
附錄13 漢黃芩素抑制IL-1+6刺激9小時所誘導之STAT3磷酸化...................................71
附錄14 gp130-STAT3-SOCS3訊號傳導途徑..................................................72

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