臺灣博碩士論文加值系統

摘 要
本研究目的是探討不同孔洞結構經genipin交聯的去細胞牛心包膜做為人工細胞外間質其體外性質與體內修復與再生的情形。
本研究分為兩部分，第一部份是利用去細胞、醋酸膨潤和膠原酵素處理的方法處理牛心包膜，以製備不同孔洞結構的牛心胞膜，並針對不同孔洞結構的牛心包膜做體外的測試，探討經不同處理具有不同孔洞結構的牛心包膜在生化性質、變性溫度和機械強度上的差異。另外，我們也進行體外抗酵素分解的實驗，預估材料在體內分解的情形。
第一部份的結果顯示，牛心包膜經去細胞、醋酸處理及酵素處理後，具有不同的孔洞結構。經去細胞後的牛心包膜，因牛心包膜內原有的細胞被去除而留下一自然的孔洞結構。而牛心包膜經醋酸處理後，因醋酸會使組織內膠原蛋白上的胺基帶正電而排斥，所以牛心包膜表面緻密的的結構會變鬆散，在牛心包膜內也會形成較大的孔洞，整體的孔隙度明顯增加。而經酵素處理後，因酵素對膠原蛋白的破壞，使得牛心包膜表面有明顯的孔洞生成，整體的孔隙度又高於醋酸處理的牛心包膜。
而經由生化分析鑑定的結果證實，牛心包膜經去細胞、醋酸處理及酵素處理後可改變牛心包膜的孔洞結構，但牛心包膜仍保留原有的細胞外間質成份。而由體外抗酵素分解的實驗我們發現，經醋酸處理的牛心包膜因膠原蛋白上的胺基帶正電排斥而膨潤，使的酵素較易攻擊，而經酵素處理的牛心包膜其膠原蛋白被膨潤並被酵素破壞，因此在抗酵素分解實驗中被分解的程度最高。
第二部分的實驗是把不同孔洞結構的牛心包膜植入老鼠背部，觀察細胞遷徙進入人工細胞外間質和組織再生的情形。以比較不同孔洞結構的牛心包膜作為人工細胞外間質的優缺點。
由實驗結果我們發現，牛心包膜在植入3天後免疫細胞主要是由牛心包膜表面滲入牛心包膜內，而牛心包膜經酵素處理後其表面的孔洞，使免疫細胞進入牛心包膜內的深度明顯高於去細胞和醋酸處理的牛心包膜。而牛心包膜在植入4週後，免疫細胞可從牛心包膜表面和尾端進入牛心包膜內。在表面部分，因醋酸處理後的牛心包膜其表面膠原蛋白纖維束結構較鬆散，在植入4週後有明顯被分解的現象，此時醋酸處理的牛心包膜具有相似於酵素處理後牛心包膜的表面孔洞結構，因此免疫細胞在植入4週後，由牛心包膜表面滲入的深度明顯高於去細胞的牛心包膜。在尾端部分，因醋酸處理和酵素處理的牛心包膜其內部的孔洞明顯高於去細胞的牛心包膜，因此免疫細胞由醋酸處理和酵素處理牛心包膜尾端進入的深度明顯高於去細胞的牛心包膜。
另外，由觀察牛心包膜植入後組織再生與修復的情形我們發現，醋酸處理和酵素處理的牛心包膜在植入後，新生組織包括新生膠原蛋白和新生葡萄糖胺聚合醣分佈的深度明顯高於去細胞的牛心包膜，因此我們推測免疫細胞進入的深度影響了組織修復與再生的範圍。另外我們也發現，孔隙度的高低影響了牛心包膜在植入後分解的速率，因為我們可觀察到去細胞的牛心包膜(孔隙度為63.4%)在植入後沒有明顯被分解的現象，而醋酸處理(孔隙度為90.5%)的牛心包膜其孔隙度高於去細胞的牛心包膜，在植入後有明顯分解的現象。
　　而牛心包膜在植入後分解的速率，會影響到組織修復完成的速率，因我們可觀察到經酵素處理的牛心包膜其分解速率快，牛心包膜完全被新生組織所取代，而經醋酸處理的牛心包膜因分解較慢所以在組織新生的區域內還有免疫細胞聚集以分解牛心包膜。
　　由動物實驗的結果證實，牛心包膜經酵素處理後其孔洞結構的改變會加速組織再生與修復的速率。因此牛心包膜經過酵素處理後更適合做為一人工細胞外間質。

目錄
內容 頁數
摘要----------------------------------------------------------------------------------I目錄------------------------------------------------------------------------------III
圖索引--------------------------------------------------------------------------VI
第一章 緒論
1-1 組織工程 1
1-2 細胞外間質 1
1-3 人工細胞外間質 4
1-4 天然生物組織材料 4
1-5 交聯劑 4
1-6 去細胞 6
1-7 孔洞結構 7
1-8 研究動機與目的 7
第二章 不同孔洞結構牛心包膜的製備與體外性質評估
2-1 研究目的 9
2-2 材料與方法 9
2-2-1 去細胞 10
2-2-2 醋酸處理 11
2-2-3 酵素處理 11
2-2-4 組織切片觀察 12
2-2-5 厚度量測 13
2-2-6 孔洞大小量測 13
2-2-7 SEM觀察 14
2-2-8 孔隙度測試 15
2-2-9 特殊組織切片染色 16
2-2-10 定量葡萄糖胺聚合醣 16
2-2-11 變性溫度 17
2-2-12 機械性質 17
2-2-13 體外抗酵素分解 19
2-3 實驗結果與討論 19
2-3-1 組織厚度 19
2-3-2 組織切片觀察 21
2-3-3 孔洞大小量測結果 23
2-3-4 表面形態分析結果 24
2-3-5 孔隙度量測結果 27
2-3-6 特殊組織切片染色結果 28
2-3-6-1 Masson`s 染色 28
2-3-6-2 Elastic Van Gieson (EVG) 染色 30
2-3-6-3 Safranin O 染色 32
2-3-7 葡萄糖胺聚合醣定量的結果 34
2-3-8 變性溫度測試結果 34
2-3-9 機械性質測試結果 35
2-3-10 體外抗酵素分解實驗結果 37
2-4 結論 39
第三章 不同孔洞結構牛心包膜在動物體內組織再生與修復評估
3-1 研究目的 40
3-2 材料與方法 40
3-2-1 試片的準備和滅菌 40
3-2-2 動物實驗 41
3-2-3 組織切片觀察 30
3-2-4 特殊組織切片染色 44
3-2-5 機械性質 44
3-2-6 鈣含量測定 45
3-3 實驗結果與討論 45
3-3-1 組織切片觀察免疫細胞進入牛心包膜的情形 45
3-3-2 組織切片觀察組織再生的情形 51
3-3-3 特殊組織切片染色的結果 59
3-3-3-1 Masson`s 染色 59
3-3-3-2 Safranin O 染色 62
3-3-4 機械性質測的結果 65
3-3-5 鈣含量測試的結果 66
3-4 結論 67
參考文獻 71

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