臺灣博碩士論文加值系統

膠原蛋白（Collagen），是多細胞動物體內含量最多的蛋白質，幾乎存在於所有組織中。膠原蛋白被廣泛應用在醫學上之生醫材料，如組織生長、燒燙傷敷料、傷口癒合等。本研究的目標在於探討膠原蛋白的型態，和glycosaminoglycan（GAG）對於骨母細胞貼附、生長與功能的影響。我們在tissue culture polystyrene（TCPS）表面上，塗佈膠原蛋白（soluble collagen）、纖維狀膠原蛋白（fibrous collagen）以及在高溫下變性膠原蛋白（denatured collagen），再將骨母細胞接種在上面，觀察細胞在不同表面上的貼附量、細胞型態、生長情況，以及功能表現。我們發現，塗佈上soluble collagen與fibrous collagen的表面，對於細胞貼附及生長效果比denatured collagen表面好，同時其鹼性磷酸酶（Alkaline Phosphatase）的活性也比較大。此外，我們也發現將膠原蛋白與肝素（heparin）之混合液塗佈在TCPS上，當肝素濃度為0.02 mg / mL時，對於細胞貼附的促進效果最佳，並且混合液會隨著肝素濃度的增加，細胞數有減少的趨勢。

Collagen and glycosaminoglycans are the major components of extracellular matrix, and are important in tissue formation and tissue engineering. This study was proposed to investigate the effects of conformation of type I collagen, and the effects of glycosaminoglycan on adhesion, proliferation and function of human osteosarcoma MG-63. Cells were seeded on TCPS plates coated with soluble collagen, fibrous collagen and heat-denatured collagen, or mixture of collagen and heparin. The attachment, morphology, growth and functions of cells were examined. We found that soluble collagen and fibrous collagen promoted cell attachment and growth more than denatured collagen. Cells cultured on soluble collagen coated plate had higher alkaline phosphatase activity than on the other substrates. In addition, we found that the plate coated with mixture of 0.02 mg/mL heparin and collagen supported better cell adhesion than the substrates coated with higher heparin concentration.

中文摘要 I
Abstract II
目錄 III
圖目錄 VI
中英文對照 XII

第一章 緒論 1
1.1 研究背景 1
1.2 膠原蛋白簡介 3
1.3 骨母細胞(osteoblast)簡介 6
1.4 研究動機 9
1.5 研究架構 10
1.6 研究目的 11
第二章 實驗藥品、儀器與方法 12
2.1 實驗藥品 12
2.1-1 細胞培養 12
2.1-2 RT-PCR 13
2.1-3 第一型與第二型膠原蛋白濃度測定 14
2.2 實驗儀器 14
2.3 溶液配製 16
2.4 實驗方法 18
2.4-1 蛋白質薄膜製備 18
2.4-1-1 不同型態膠原蛋白製備 18
2.4-1-2 膠原蛋白與肝素混合液製備 18
2.4-1-3 蛋白質薄膜塗佈 19
2.4-1-4 薄膜表面分析 19
2.4-1-5 蛋白質薄膜定量 20
2.4-2 細胞培養 20
2.4-3 細胞佈殖 21
2.4-4 細胞數測定 21
2.4-5 鹼性磷酸酶活性測定 22
2.4-6 RT-PCR 23
2.4-6-1 萃取RNA 23
2.4-6-2 反轉錄（Reverse transcription；RT） 24
2.4.6-3 Polymerase chain reaction（PCR） 24
2.6-4-4 DNA電泳 25
2.6-4-5 統計分析軟體 26
第三章 結果與討論 27
3.1 膠原蛋白的吸附 27
3.2 在TCPS上塗佈不同形態的膠原蛋白，對骨母細胞貼附、生長及基因表現 31
3.2-1 骨母細胞貼附在塗佈不同形態膠原蛋白的TCPS上 31
3.2-2 不同型態的膠原蛋白對於鹼性磷酸酶活性的影響 36
3.2-3 骨母細胞在不同型態的膠原蛋白上之生長、鹼性磷酸酶活性與基因表現 39
3.3 在TCPS上塗佈膠原蛋白與肝素和膠原蛋白混合液，對骨母細胞貼附、生長及基因表現 42
3.3-1 骨母細胞貼附在塗佈不同濃度的肝素和膠原蛋白之混合液的表面 42
3.3-2 骨母細胞在不同濃度的肝素和膠原蛋白之混合液的表面上之生長與鹼性磷酸酶活性 45
第四章 結論 84
4.1 結論 84
4.2 未來研究方向 86
參考目錄 88

圖目錄

圖1-1 具有週期性的膠原蛋白纖維之軸向結構 5
圖1-2 形成纖維狀膠原蛋白示意圖 5
圖3-1 TCPS表面塗佈上不同型態膠原蛋白 48
圖3-2 TCPS表面塗佈上不同型態膠原蛋白 48
圖3-3 TCPS表面塗佈上不同濃度膠原蛋白和肝素混合液 49
圖3-4 Mica塗佈上soluble collagen之AFM高度圖。 49
圖3-5 Mica塗佈上soluble collagen之AFM相圖。 50
圖3-6 Mica塗佈上soluble collagen之AFM高度分析圖。 50
圖3-7 Mica表面塗佈上fibrous collagen之AFM高度圖。 51
圖3-8 Mica表面塗佈上fibrous collagen之AFM相圖。 51
圖3-9 Mica塗佈上fibrous collagen之AFM高度分析圖。 52
圖3-10 Mica塗佈上denatured collagen之AFM高度圖。 52
圖3-11 Mica塗佈上denatured collagen之AFM相圖。 53
圖3-12 Mica塗佈上denatured collagen之AFM高度分析圖。 53
圖3-13 在有血清的培養液中，材料表面塗佈上不同型態膠原蛋白，經培養1、2、4及24小時後測得之細胞數 54

圖3-14 在無血清的培養液中，材料表面塗佈上不同型態膠原蛋白，經培養1、2、4及24小時後測得之細胞數 54
圖3-15 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過1小時的培養後之細胞型態 55
圖3-16 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過2小時的培養後之細胞型態 56
圖3-17 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過4小時的培養後之細胞型態 57
圖3-18 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過24小時的培養後之細胞型態 58
圖3-19 在無血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過1小時的培養後之細胞型態 59
圖3-20 在無血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過2小時的培養後之細胞型態 60
圖3-21 在無血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過4小時的培養後之細胞型態 61
圖3-22 在無血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過24小時的培養後之細胞型態 62


圖3-23 在有血清的培養液中，材料表面塗佈上不同型態膠原蛋白，經培養4和24小時後測得之細胞數) 63
圖3-24 在無血清的培養液中，材料表面塗佈上不同型態膠原蛋白，經培養4和24小時後測得之細胞數 63
圖3-25 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經培養4和24小時後測得之鹼性磷酸磷量 64
圖3-26 在無血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經培養4和24小時後測得之鹼性磷酸磷量 64
圖3-27 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經培養4和24小時後測得之鹼性磷酸磷活性) 65
圖3-28 在無血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經培養4和24小時後測得之鹼性磷酸磷活性 65
圖3-29 在有血清的培養液中，材料表面塗佈上不同型態膠原蛋白，經培養1天、3天和7天後測得之細胞數 66
圖3-30 在有血清的培養液中，材料表面塗佈上不同型態膠原蛋白，經培養4和24小時後測得之鹼性磷酸磷活性 66
圖3-31 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經培養1天後之細胞型態 67


圖3-32 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經培養3天後之細胞型態 68
圖3-33 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經培養7天後之細胞型態 69
圖3-34 骨母細胞( 98代 )在有血清的培養液中，經過培養7天後，對於GAPDH、alkaline phosphatase( ALP )、osteopontin、type I collagen的基因表現 70
圖3-35 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過培養7天後，ALP 、osteopontin及type I collagen（col I）對GAPDH之相對強度。（第98代骨母細胞） 70
圖3-36 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過培養7天後，ALP 、osteopontin及type I collagen（col I）對GAPDH之相對強度（第99代骨母細胞） 71
圖3-37 在有血清的培養液中，並於材料表面塗佈上不同型態膠原蛋白，經過培養7天後，ALP 、osteopontin及type I collagen（col I）對GAPDH之相對強度（第95代骨母細胞） 71
圖3-38 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養4和24小時後測得之細胞數 72

圖3-39 在無血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養4和24小時後測得之細胞數 72
圖3-40 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養4和24小時後測得之鹼性磷酸酶活性 73
圖3-41 在無血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養4和24小時後測得之鹼性磷酸酶活性 73
圖3-42 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養1小時後之細胞型態 74
圖3-43 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養2小時後之細胞型態 75
圖3-44 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養4小時後之細胞型態 76
圖3-45 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養24小時後之細胞型態 77
圖3-46 在無血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養1小時後之細胞型態 78

圖3-47 在無血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養2小時後之細胞型態 79
圖3-48 在無血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養4小時後之細胞型態 80
圖3-49 在無血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養24小時後之細胞型態 81
圖3-50 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養1天和3天後測得之細胞數 82
圖3-51 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養1天和3天後測得之鹼性磷性酶活性 82
圖3-52 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養1天和3天後測得之細胞數 83
圖3-53 在有血清的培養液中，並於材料表面塗佈上膠原蛋白及肝素與膠原蛋白之混合液，經培養1天和3天後測得之鹼性磷酸酶活性 83

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