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研究生:古健良
研究生(外文):Ku, Chien-Liang
論文名稱:探討 AMPK訊息參與三維培養提昇間葉基質幹細胞免疫調節活性的機轉
論文名稱(外文):Explore the Mechanism of AMPK Signals Involved in Three-Dimensional Culture Promoting the Immune Regulatory Activity of Mesenchymal Stem Cells
指導教授:周秀慧周秀慧引用關係
指導教授(外文):Chou, Shiu-Huey
口試委員:羅正汎沈家瑞周秀慧
口試委員(外文):Lo, Jeng-FanShen, Chia-RuiChou, Shiu-Huey
口試日期:2020-07-29
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
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:2020
畢業學年度:108
語文別:中文
論文頁數:119
中文關鍵詞:間葉基質幹細胞羊水幹細胞三維培養單磷酸腺苷活化蛋白質激酶免疫 抑制鈣離子鈣調蛋白依賴性蛋白激酶激酶 2
外文關鍵詞:mesenchymal stem cellsAFSCthree-dimensional cultureAMPKimmune suppressioncalcium ionCaMKK
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目錄……………………………………………………………………………………...I
附表目錄………………………………………………………………………………IV
附圖目錄……………………………………………………………………………….V
英文縮寫表………………………………………………………………………….VIII
摘要…………………………………………………………………………………….X
英文摘要………………………………………………………………………………XI
第一章 研究背景……………………………………………………………………….1
第一節 間葉基質幹細胞的特性與應用…………………………………………….1
1.1.1間葉基質幹細胞的特性與來源……………………………………………..1
1.1.2 MSCs的免疫調節活性……………………………………………………...2
1.1.3 圍產期幹細胞的特性……………………………………………………….4
1.1.4 圍產期幹細胞的應用……………………………………………………….6
第二節 幹細胞的三維培養………………………………………………………….8
1.2.1 MSCs的應用瓶頸…………………………………………………………...8
1.2.2三維培養的特色與培養方法………………………………………………..9
1.2.3 MSCs三維培養研發的回顧……………………………………………….11
第三節 三維培養對MSCs免疫調節活性的影響………………………………...13
第四節 AMPK對免疫調節活性的影響…………………………………………..14
1.4.1 AMPK的簡介………………………………………………………………14
1.4.2 AMPK的訊息活化機制……………………………………………………15
1.4.3 AMPK對免疫細胞活性的影響……………………………………………16
1.4.4 AMPK對MSCs活性的影響……………………………………………….17
第二章 研究目的與重要性…………………………………………………………...19
第三章 實驗材料與方法……………………………………………………………...22
第一節 實驗材料…………………………………………………………………...22
3.1.1小鼠羊水間葉性幹細胞株…………………………………………………22
3.1.2小鼠胎盤間葉性幹細胞株…………………………………………………22
3.1.3 實驗藥品、培養基及各類試劑……………………………………………22
第二節 設備儀器…………………………………………………………………...30
第三節 實驗方法…………………………………………………………………...32
3.3.1 羊水、羊膜及胎盤間葉幹細胞之二維培養………………………………32
3.3.2 幾丁質聚醣 (Chitosan membrane, CS)薄膜製備………………………...32
3.3.3 羊水、羊膜及胎盤間葉幹細胞之三維培養……………………………….32
3.3.4三維培養之羊水、羊膜及胎盤間葉幹細胞的球體大小、細胞擴增與生存
率的評估………………………………………………………………………….33
3.3.5羊水、羊膜及胎盤間葉幹細胞之蛋白質表現量…………………………33
3.3.6以反轉錄聚合連鎖反應(RT-PCR)評估羊水及胎盤間葉幹細胞免疫抑制
基因之表現……………………………………………………………………….34
3.3.7 統計分析…………………………………………………………………...36
第四章 結果…………………………………………………………………………...37
第一節 三維培養可提升間葉幹細胞的免疫調節分子基因的表現與AMPK的磷
酸化………………………………………………………………………………….37
4.1.1 羊水幹細胞二維培養的建立與其表面MSCs相關分子的分析………...37
4.1.2 三維培養方式對不同來源之間葉幹細胞的免疫調節分子與AMPK磷酸
化的影響………………………………………………………………………….38
4.1.3 不同的三維培養方法對羊水間葉幹細胞免疫調節分子的基因與AMPK
磷酸化的影響…………………………………………………………………….40
III
第二節 AMPK訊息的活化參與三維培養提升羊水間葉幹細胞免疫調節分子基
因的表現………………………………………………………………………….....41
4.2.1 AICAR可以提升二維培養時羊水間葉幹細胞中免疫調節分子基因的表
現………………………………………………………………………………….42
4.2.2 Compound C抑制倒掛三維培養提升羊水間葉幹細胞中免疫調節分子基
因的表現………………………………………………………………………….43
第三節 三維培養提升羊水間葉幹細胞之免疫調節因子表現活性是透過鈣離子
活化AMPK的訊息途徑……………………………………………………………45
4.3.1 三維培養對羊水間葉幹細胞中AMPK上游訊息的影響………………..45
4.3.2 三維培養對羊水間葉幹細胞中AMPK的下游訊息的影響…………….50
第五章 討論…………………………………………………………………………...52
第六章 總結…………………………………………………………………………...60
第七章 參考文獻……………………………………………………………………...62
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