臺灣博碩士論文加值系統

　心肌細胞一般被認為是人體中最具代表性，無法增殖、再生、分化的細胞，一旦心肌細胞受損或壞死而失去功能時，成熟的心肌細胞難以再生。心臟功能的重建，除了心臟移植及心室輔助器兩種方式，還包括了以再生心肌細胞的方式來重建受損心肌細胞。
　有不少研究採用五氮胞&;#33527;對間質幹細胞做化學的誘導，研究結果顯示五氮胞&;#33527;可用來誘導間質幹細胞分化成肌性細胞，誘導後可觀察到自發性的跳動並測出相類似於心肌細胞的動作電位。膠原蛋白為細胞外基質主要成分之一，俱有維持細胞形狀、調節組織形態並作為細胞貼附支架等功能，透明質酸對細胞的聚集、遷移、增生及分化有重大的影響，俱有極佳的生物相容性及生物可分解性，故常被用作組織工程支架。
　本研究第一階段在製備研究不同分子量之透明質酸/膠原蛋白基材，未交聯之透明質酸/膠原蛋白基材浸於二次水中即無法成型，經過交聯後之透明質酸/膠原蛋白基材，於濕潤的環境下，仍可保持其原本形狀，其含水量皆可高達95%以上，孔隙率60~80%。透明質酸/膠原蛋白基材之機械性質結果表示，交聯後之複合材料在固定應力施加下，其伸長量及楊氏系數隨著透明質酸含量的增加而增加，顯示透明質酸濃添加越多，複合材料之機械強度越高，較易因應所承受之應力產生相當變形量以符合欲填補之組織缺陷。
　本研究第二階段以膠原蛋白/透明質酸混合仿生材做為「支架」，以間質幹細胞為「細胞」，五氮胞&;#33527;則為刺激幹細胞分化之「訊息誘導因子」，利用此心補片植入動物心肌損傷區域進行心肌損傷修復評估，以期更加了解臨床試驗上之應用並有效的觀察細胞之分化。由結果發現種於基材中的細胞的心肌細胞特異基因(cTnIl、β-MHC、α-actin) 表現量在前期遠高於僅以五氮胞&;#33527;誘導之組別。隨著培養天數的增加，發現有未添加透明質酸之組別其cTnI、β-MHC、α-actin基因表現量驟降，但在添加透明質酸之組別，其cTnI、β-MHC、α-actin基因表現顯著高於其他組別，由此可看出透明質酸對細胞分化的重要性。
　第三階段則為心肌梗塞動物模型的建立，採用結紮心臟左前降支血管來使左心室前璧尖端肌肉產生壞死現象。由組織切片可清楚觀察到術後之大白鼠左心室已有部分心肌被纖維細胞取代，細胞排列凌亂及細胞分布不密集，由此證明利用縫線結紮心臟左前降支，可對心肌造成損傷。將種植幹細胞之膠原蛋白/透明質酸基材經誘導後，植入於大鼠心肌梗塞處，植入4週後雖未達到完整修復之效果，然於靠近中心部分的心肌組織，已呈現出接近正常的心肌組織，同時細胞與細胞外基質排列較為緻密，且組織最外圍亦呈現平整結構，顯示膠原蛋白/透明質酸基材於心肌缺損修復上具有發展之潛力。

As well known, cardiomyocytes and neural cells are un-proliferative cells in human. Especially, when the cardiomyocytes damaged, necrotized and loosed its function, the heart is difficult to recover to its original function. To date, in clinical practice, the remedy of this damaged heart is by heart transplantation or heart assist devices. Besides, by implanting regenerative cardiomyocytes into the defect area is now acquiring more attentions from many recent researchers. 5-azacytidine is used to differentiate the mesenchymal stem cells (MSCs) to cardiomyocyte-like cells in these researches. These cardiomyoctye-like cells reveal the spontaneous heart-beat behaviors and with action potentials as normal cardiomyocytes. Type I collagen is the main composition of extracellular matrix. It provides many cellular functions, including providing scaffolds for cells adhesion, migration, differentiation and proliferation. Hyaluronic acid, also possesses biocompatible and biodegradable properties, and used to prepare cell-cultural substrates in tissue engineering applications. The aims (or works) of this study are to prepare 3-D mimetic scaffold (cardiopatch) which be able to provide a natural like micro environment for MSCs growth, to evaluate a chemical stimulant which act as a signal transduction factor, 5-azacytidine is added into the scaffold or medium to stimulate MSCs differentiate to the cardiomyocytes, and to establish animal model with myocardial injury. According to the analysis of water content, porosity and mechanical properties of collagen/hyaluronan composite scaffold, the group composite scaffold (collagen I : HA of 7:1.5) has the maximum elongation. The effect of 5-azacytidine used to induce differentiation of mesenchymal stem cells into cardiomyocyte was positively confirmed by real-time PCR. The results showed that mesenchymal stem cells seeded in composite scaffold expressed higher value of cardiomyocyte specific genes (such as cTnI, β-MHC and α-actin) than the group of cells without using any scaffold but only 5-azacytidine. During the buildup of animal model with myocardial damages, operation was performed to suture the left anterior descending artery and it resulted dark red color change on the distal end of myocardium, biopsy observation also reported that part of myocardium have been replaced with fibroblast with mesenchymal cells alignment or distribution. This proves that by suturing the left anterior descending artery can cause damages to myocardium. The animal study demonstrated that collagen/HA composite scaffolds have the potential for cardiac regeneration applications.

目錄
誌謝 2
中文摘要 4
英文摘要 6
目錄 8
圖目錄 10
表目錄 12
第一章 緒論 13
1.1　心臟衰竭 13
1.2　心肌組織工程 17
1.2.1　幹細胞來源 20
1.2.2　訊息因子 26
1.2.3　支架 28
1.3　心肌特異基因表現 31
1.4　研究背景與目的 33
第二章　實驗材料與儀器設備 35
2.1　實驗材料 35
2.2　儀器設備 37
第三章　研究方法 38
3.1　第一型膠原蛋白萃取及鑑定 38
3.1.1　第一型膠原蛋白萃取 38
3.1.2　第一型膠原蛋白之鑑定 38
3.1.3　第一型膠原蛋白濃度測定 39
3.2　基材製備 39
3.3　基材物理性質分析 40
3.3.1　含水量測試 40
3.3.2　孔隙率測試 40
3.3.3　機械性質測試 40
3.4　間質幹細胞之分離培養 41
3.4.1　間質幹細胞的萃取 41
3.4.2　間質幹細胞的培養 41
3.4.3　間質幹細胞於基材三維培養模式 41
3.4.4　反轉錄聚合&;#37238;鏈反應分析 42
3.5 動物實驗 44
3.5.1心肌損傷之動物模型建立 44
3.5.2 基材植入心肌損傷動物實驗 47
3.5.3 組織切片染色 48
第四章 結果與討論 49
4.1 第一型膠原蛋白濃度測定 49
4.2膠原蛋白/透明質酸複合基材分析 50
4.2.1微結構觀察 50
4.2.2含水量測試 51
4.2.3孔隙率測試 52
4.2.4機械性質測試 53
4.3 間質幹細胞萃取及鑑定分析 56
4-3-1 間質幹細胞分離培養 56
4.3.2 間質幹細胞鑑定分析 57
4.4反轉錄聚合&;#37238;鏈反應分析 59
4.5心肌梗塞動物模型 62
4.6動物植入實驗 64
第五章 結論 66
參考文獻 68

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