開發體外培養子宮內膜類組織應用於子宮沾黏症候群相關研究

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姓名	楊勳岳(Syun-Yue Yang) 查詢紙本館藏	畢業系所	生物醫學工程研究所
論文名稱	開發體外培養子宮內膜類組織應用於子宮沾黏症候群相關研究 (Development of In-vitro Artificial Endometrial Tissues for Applications in Intrauterine Adhesions)
檔案	[Endnote RIS 格式] [Bibtex 格式] [相關文章] [文章引用] [完整記錄] [館藏目錄] 至系統瀏覽論文 (2029-7-31以後開放)
摘要(中)	子宮內膜對女性的生殖健康至關重要，由幾種不同類型的上皮細胞和基質細胞組成，上皮細胞與基質細胞之間的細胞通訊對於子宮內膜的正常功能至關重要。子宮沾黏症候群（Intrauterine Adhesions，IUA）主要發生在刮宮術（Dilation and Curettage，D&C）後，也可能在子宮動脈栓塞手術後發生。這主要是由於子宮內膜受損，導致基質細胞異常增生，使內膜之間產生組織沾黏。過去，治療子宮沾黏通常使用子宮鏡和宮內節育器以及激素刺激來治療；近年來，研究發現使用水凝膠作為術後預防以及細胞治療的方式具有潛力。本研究著重於水凝膠治療效果和子宮內膜類組織的開發。隨著類器官培養技術的快速發展，許多體外模擬的類器官可以呈現體內器官的部分功能。在第一部分，本研究開發了一種方法，通過結合人類子宮內膜上皮細胞（human endometrial epithelial cells，hEEC）和人類子宮內膜基質細胞（human endometrial stromal cells，hESC）以及3D 列印技術，利用體外培養技術培育子宮內膜類組織。此研究以膠原蛋白作為支架，基質細胞預先與膠原蛋白墨水（Collgel®）混合，然後通過3D 列印出圓盤，將上皮細胞種植在支架表面，形成人工子宮內膜類組織（artificial Endometrial Tissues，AEC）。後續對AEC進行染色及觀察細胞在支架內的生長形態，旨在建立體外子宮模型，未來可用於觀察子宮沾黏的體外模型以及藥篩平台的測試。第二部分則進行動物實驗。實驗將小鼠的子宮通過針頭刺傷模擬子宮受損，然後注射水凝膠，觀察術後預防的效果。這一階段旨在比較動物模型與體外模型之間的差異，並評估水凝膠的治療效果。
摘要(英)	The endometrium is crucial for women′s reproductive health, consisting of different types of epithelial and stromal cells. Communication between epithelial and stromal cells is essential for the physiological functions of the endometrium. Intrauterine adhesions (IUA) commonly occur after dilation and curettage (D&C) procedures and can also follow uterine artery embolization. This condition arises from endometrial damage leading to abnormal proliferation of stromal cells, resulting in tissue adhesions between the endometrial layers. Historically, IUA treatments have involved hysteroscopy, intrauterine devices, and hormone therapy. Recently, research has explored the use of hydrogels for postoperative prevention and cell therapy for treatment.This study focuses on the effectiveness of hydrogel treatment and the development of endometrial-like tissues. With the rapid advancement of organoid culture techniques, many in vitro organoids can mimic some functions of in vivo organs. In the first part of this study, we developed a method combining human endometrial epithelial cells (hEEC) and human endometrial stromal cells (hESC) with 3D printing technology to cultivate endometrial-like tissues in vitro. Collagen was used as a scaffold, with stromal cells pre-mixed with collagen ink (Collgel®) and 3D printed into discs. Epithelial cells were then seeded on the scaffold surface to form artificial endometrial tissues (AEC). These AECs were subsequently stained and observed to examine cell growth patterns within the scaffold. This phase aims to establish an in vitro uterine model, which can later be used to observe IUA models and test drug screening platforms.The second part of the study involves animal experiments. The uteri of mice were damaged using needles to simulate endometrial injury, followed by hydrogel injection to observe the effectiveness of postoperative prevention. This phase aims to compare differences between animal models and in vitro models and to evaluate the therapeutic effects of the hydrogel.
關鍵字(中)	★ 子宮沾黏症候群 ★ 三維培養 ★ 子宮內膜類器官 ★ 3D列印 ★ 生物支架	關鍵字(英)	★ Intrauterine adhesions ★ 3D culture ★ Endometrial organoid ★ 3D printing ★ Bioscaffold
論文目次	摘要 i ABSTRACT ii 致謝 iv 目錄 vi 圖目錄 xi 表目錄 xiii 符號縮寫表 xiv 第一章研究背景暨文獻回顧 1 1-1 子宮（Uterine） 1 1-1-1月經週期（Menstrualcycle） 2 1-1-2 子宮沾黏症候群（Intrauterine Adhesions） 3 1-2 細胞培養（ Cell Culture） 4 1-2-1 三維細胞培養（3D Cell Culture） 4 1-2-2 人類子宮內膜上皮細胞（hEEC） 5 1-2-3 人類子宮內膜基質細胞（hESC） 5 1-3 類器官（Organoids） 7 1-4 3D生物列印（3D Bio-printing） 7 1-4-1 3D生物列印機（3D Bio-printer） 8 1-4-2 膠原蛋白生物墨水（Collgel® Bioink） 9 1-5 ALi培養方式（Air-liquid interface） 10 1-6 即時聚合酶連鎖反應（qPCR） 11 1-7 材料選用（Material Selection） 12 1-7-1 明膠（Gelatin） 12 1-7-2 微生物轉麩胺酸醯胺基酶（mTG） 13 1-7-3 聚丙烯酸鈉（Acrylic Sodium Salt Polymer） 13 1-8 體內IUA模型（In-vivo Animal Model） 14 第二章研究動機與實驗目的 15 2-1研究動機（Research Motivation） 15 2-2研究目的（Research Purposes） 16 第三章材料與方法 17 3-1 耗材（Consumables） 17 3-2 儀器（Instruments） 18 3-3 細胞培養（Cell Culture） 20 3-3-1細胞培養（Cell Culture） 20 3-3-2 1x PBS 溶液（1x PBS Solution） 20 3-3-3細胞繼代（Cell Subculture） 20 3-3-4人類子宮內膜異位症上皮細胞（hEEC） 21 3-3-5人類子宮內膜基質細胞（hESC） 21 3-3-6 E2和E2/P4刺激培養液配置（E2&E2/P4 Culture Medium） 22 3-4 生物3D列印（3D Bio-printing） 22 3-4-1 膠原蛋白生物墨水製備（Collgel ®） 22 3-4-2 hESC生物墨水製備（Preparation of Collgel ® with hESC） 22 3-4-3 3D列印（3D Print） 23 3-5 ALi培養系統（ALi system） 25 3-5-1 插入式細胞培養皿（Transwell） 25 3-6 生物反應器（Bioreactor） 25 3-7 體外IUA模型（In-vitro IUA model） 26 3-8 膠體內細胞固定（Collgel® Cell Fixed） 27 3-9細胞染色分析（Cell Staining Analysis） 27 3-9-1共軛焦顯微鏡（Confocal Microscopy） 27 3-9-2 細胞膜染色（Cell Membrane Stain） 28 3-9-3 細胞核和細胞骨架染色（Nucleus and Cytoskeletal Stain） 28 3-9-4細胞死活染色（Live/Dead Stain） 28 3-9-5免疫螢光染色（Pan-cytokeratin/Vimentin Stain） 29 3-10 即時聚合酶連鎖反應（qPCR） 29 3-11 水凝膠製備（Hydrogel preparation） 31 3-11-1 mTG製備（mTG preparation） 31 3-11-2 配置水凝膠（Configure hydrogel） 31 3-11-3 成膠&膨脹實驗（Gel-barrier Formation/Swelling test） 32 3-11-4 體內模型建立（In-vivo animal model） 32 第四章結果與討論 33 4-1 生物3D列印（3D bio-print） 33 4-1-1生物3D列印子宮內膜類組織（Artificial Endometrial Tissues） 33 4-1-2 3D列印膠體存活測試（Live/Dead Test） 34 4-1-3子宮內膜類組織細胞染色觀察（AEC staining） 34 4-1-3 子宮內膜類組織細胞分析（AEC Cell analysis） 35 4-2激素刺激（Hormone stimulation） 38 4-2-1 AEC細胞膜染色（AEC Cell Membrane Staining） 38 4-2-2 AEC激素刺激細胞分析（AEC Horomone-Stimulated） 38 4-2-2 AEC激素刺激對於上皮影響（Effects of epithelial on AEC） 40 4-3 ALi培養（ALi Culture） 41 4-4 ALi動態培養（ALi dynamic Culture） 44 4-4-1 ALi生物反應器架設（ALi Bioreactor Setup） 44 4-4-2 ALi動態培養細胞分析（ALi dynamic Culture Cell Analysis） 46 4-5 體外IUA模型（In-vitro IUA Model） 48 4-6 即時聚合酶連鎖反應（qPCR） 49 4-7水凝膠成膠和膨脹測試（Gel Formation/Swelling test） 51 4-8體內動物模型（In-vivo model） 52 第五章結論 55 第六章參考文獻 56
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指導教授	陳靖昀許世典(Ching-Yun Chen Shih-Tien Hsu)	審核日期	2024-7-29
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