體外仿生心臟衰竭三維模型研究

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姓名

蕭廷儒(Ting-Ru Siao) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

體外仿生心臟衰竭三維模型研究
(Investigation of In Vitro Bionic 3D Heart Failure Model)

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摘要(中)

心血管疾病當前名列全球三大死因之一，其中心力衰竭造成的死亡率偏高。因此，開發準確模擬心臟衰竭的研究模型是相當重要的。在人體內的細胞行為會與其微環境動態地相互作用，因此在實驗環境中重現這種相互作用至關重要。在這項研究中，採用甲基丙烯酸酐化豬明膠構建的三維支架可透過機械循環拉伸和壓縮來複製體內微環境。機械條件為10%應變和1.5 Hz頻率。本研究調查了三維支架內機械循環負荷對AC16人類心肌細胞的影響。研究結果表明，在動態三維支架中培養的AC16細胞在第3天和第7天表現出F-肌動蛋白、TGF-β 和YAP的表達量升高，證實了3D仿生動態培養促進細胞生長，而在動態三維支架中培養的AC16細胞在第3天和第7天心臟衰竭相關疾病BNP、CRP、desmin、myosin、TNNI3的表現量也是升高，並從細胞衍生的外泌體中發現miRNA對細胞的影響。

摘要(英)

Cardiovascular diseases presently rank among the top three leading global causes of mortality, with heart failure representing a significant contributor to this high mortality rate. Consequently, it is imperative to develop research models that accurately simulate heart failure. Cellular behavior within the human body dynamically interacts with its microenvironment, making it essential to recreate this interaction in experimental settings. In this study, a three-dimensional scaffold constructed from porcine gelatin methacryloyl was employed to replicate the in vivo microenvironment through mechanical cyclic stretching and compression. These mechanical conditions involved a 10% strain and a 1.5 Hz frequency. The research investigated the impact of mechanical cyclic loading within the three-dimensional scaffold on AC16 human cardiomyocyte cells. The findings reveal that AC16 cells cultured within dynamic three-dimensional scaffolds exhibited heightened expression of F-actin, TGF-β, and YAP at three and seven days, providing confirmation that 3D biomimetic dynamic culture fosters cellular growth. In the dynamic three-dimensional scaffold, the AC16 cells cultured showed an increase in the expression levels of BNP, CRP, desmin, myosin, and TNNI3, which are associated with heart failure, on the 3rd and 7th days. Additionally, the impact of miRNA on the cells was observed in the exosomes derived from the cells.

關鍵字(中)

★ 心臟衰竭
★ 三維模型研究

關鍵字(英)

★ heart failure
★ 3D Heart Failure Model

論文目次

中文摘要 i
Abstract ii
目錄 iii
圖目錄 vi
表目錄 vii
第一章緒論 1
1-1 心臟衰竭 1
1-2 仿生模型 1
1-3 微環境 2
1-4 3D支架材料 2
1-4-1 膠原蛋白 2
1-4-2 海藻酸鹽 2
1-4-3 甲基丙烯酸酐化明膠 3
1-5 生物製造 3
1-6 動態培養 3
1-7 外泌體 4
1-8 次世代定序 4
1-9 研究動機與目的 5
第二章材料與方法 6
2-1 實驗方法 6
2-1-1 細胞培養 6
2-1-2 製備甲基丙烯酸酐化豬明膠(GelMA) 6
2-1-3 甲基丙烯酸酐化豬明膠物化性質分析 7
2-1-3-1 流變儀分析 7
2-1-3-2 掃描式電子顯微鏡(SEM) 7
2-1-3-3 傅立葉紅外光譜(FTIR) 8
2-1-3-4 核磁共振光譜(NMR) 8
2-1-4 細胞機械循環擠壓 8
2-1-5 細胞毒性分析 9
2-1-6 細胞型態及免疫螢光染色 9
2-1-7 鈣離子濃度測定 12
2-1-8 外泌體純化 12
2-1-9 奈米顆粒追蹤分析實驗(NTA) 13
2-1-10 穿透式電子顯微鏡(TEM) 13
2-1-11 流式細胞技術(flow cytometry) 14
2-1-12 西方墨點法 14
2-1-13 NGS數據分析 14
2-2 實驗材料 15
2-2-1 細胞培養 15
2-2-2 甲基丙烯酸酐化豬明膠材料 16
2-2-3 3D模具 16
2-2-4 機械循環擠壓 16
2-2-5 螢光染色實驗 16
2-2-6 鈣離子濃度測定 17
2-2-7 外泌體純化 17
2-2-8 奈米顆粒追蹤分析實驗 17
2-2-9 流式細胞技術 17
2-2-10 西方墨點法 18
2-2-11 NGS實驗 19
第三章實驗流程 20
第四章實驗結果 21
4-1 甲基丙烯酸酐化豬明膠物化性質分析 21
4-1-1 流變儀分析 21
4-1-2 掃描式電子顯微鏡(SEM) 22
4-1-3 傅立葉紅外光譜(FTIR) 23
4-1-4 核磁共振光譜(NMR) 25
4-2 支架模具、支架外觀與擠壓儀器 26
4-3 細胞毒性分析 27
4-4 AC16細胞機械循環擠壓下的免疫螢光染色 29
4-5 鈣離子濃度測定 37
4-6 外泌體特徵 38
4-7 外泌體基因篩選 40
4-8 經由機械循環擠壓細胞衍生的外泌體和靜態培養有交集的差異表達基因 41
4-9 KEGG途徑富集分析 42
4-10 差異表現基因的訊號通路分析 42
第五章討論與結論 44
5-1 討論 44
5-1-1 特定條件下機械循環擠壓能使心肌細胞疾病表現量上升 44
5-1-2 探討機械循環擠壓細胞衍生的外泌體與控制組差異表達基因 44
5-2 結論 45
參考文獻 47

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指導教授

許藝瓊

審核日期

2024-8-14

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