臺灣博碩士論文加值系統

本研究的目的於比較不同材料對軟骨細胞貼附、生長及基因表現的影響。我們採用PDLLA、PLLA、PLGA及PCL四種可分解高分子材料，利用solvent-casting方法製成薄膜，培養軟骨細胞，分別於1天、7天及14天內觀察軟骨細胞於薄膜表面貼附、生長及基因表現。本研究發現在細胞生長方面，第7天時，PLGA及PDLLA有較多的細胞數，但第14天時各種高分子表面的細胞數卻沒有統計上的不同。在基因表現方面，從RNA-level觀察了第一型膠原蛋白、第二型膠原蛋白以及aggrecan的基因表現。不同材料間有很大的差異。以14天的培養期長度來看，PDLLA細胞在14天時恢復了其基因表現型，是裡面表現最好的材料。
接下來，將選擇的PDLLA高分子薄膜表面分別吸附上第一型膠原蛋白、第二型膠原蛋白、纖維網蛋白三種ECM蛋白質及聚離胺酸。於1天、4天、7天及14天觀察軟骨細胞在其薄膜上貼附、生長及基因表現。在細胞生長方面，14天時，吸附上第一型膠原蛋白、第二型膠原蛋白、纖維網蛋白的PDLLA表面有較多的細胞數，又以吸附纖維網蛋白的PDLLA薄膜上最多。而基因表現方面，各種材料表面的軟骨細胞一直到14天都能夠維持第二型膠原蛋白及aggrecan的基因表現，並沒有太多的不同。

The goal of this study was to compare the performance of chondro- cytes in adhesion, proliferation and gene expression of chondrocytes when they were cultivated on different biodegradable polymers. Four kinds of biodegradable polymers was used in this study, including PDLLA, PLLA, PLGA, and PCL, which were made by using the solvent- casting method, and chondrocytes were cultured on the polymer films. Cell adhesion on day 1, cell proliferation and gene expression on day 4 and day 7 were investigated. The results show that chondrocytes prolife- rated better on PLGA and PDLLA films in 7 days, but there are no signi- ficant differences on each film for 14 days. The gene expression on diffe- rent polymer films showd difference. We suggest the PDLLA is most suitable for chondrocyte culture, since the gene expression of chondro- cytes is restored during 14 days culture period.
As a result, PDLLA films was modified by adsorption of collagen type I, collagen type II, fibronectin, and polylysine. Cell adhesion on day 1, cell proliferation and gene expression on day 4,7,14 were investigated. The results showed that collagen type I, collagen type II, and fibronectin could enhance chondrocytes proliferation during culture. The gene expression was retained during 14 days on each surface. It seems that the adsorbed proteins would not effect the gene expression of chondrocytes.

誌謝….…………………………………………………………………I
中文摘要.…………………………………………………………… III
英文摘要………………………………………………………………V
目錄……………………………………………………………………VII
圖目錄…………………………………………………………………XI
縮寫與符號說明……………………………………………………XIV
中英名詞對照………………………………………………………XVI



第一章 緒論
1.1研究背景…………………………………………………………1
1.2關節軟骨…………………………………………………………3
1.3關節軟骨的受損與修復……….…………………………………7
1.4組織工程…………………………………………………………9
1.5 研究動機……………………..………….……………………18
1.6 研究架構……………………………………………………….19



第二章 實驗藥品、儀器與方法
2.1實驗藥品……………………………..........…………….21
2.1.1 高分子薄膜製備……………………....……………………21
2.1.2細胞培養………………………………....………………….21
2.1.3 RT-PCR…………………………………………………22
2.1.4第一型及第二型膠原蛋白分離純化及濃度測定………23
2.1.5薄膜表面吸附蛋白質……………………………………23
2.1.5固定細胞及染色……………………………......……………23
2.2實驗儀器…………………………………………………………23
2.3 溶液配製……………………………………..………..…… 25
2.4實驗方法…………………………………………………………27
2.4.1可分解高分子對細胞貼附生長及基因表現影響
2.4.1.1高分子薄膜製備…………………………………27
2.4.1.1.1玻璃(培養皿)表面疏水處理……………27
2.4.1.1.2 Solvent-casting method………………28
2.4.1.2薄膜表面測試……………………………………28
2.4.1.2.1旋轉塗佈(spin coating)………………28
2.4.1.2.2接觸角(contact angle)量測……………28
2.4.1.3細胞培養 ………………………………………28
2.4.1.4細胞佈殖(seeding)……………………………29
2.4.1.5細胞數測試………………………………………29
2.4.1.6 RT-PCR…………………………………………30
2.4.1.6.1萃取RNA…………………………………30
2.4.1.6.2反轉錄(Reverse transcription)……30
2.4.1.6.3 Polymerase chain reaction…………31
2.4.1.6.4 DNA電泳……………………………32
2.4.1.6.52電泳強度分析………………………32
2.4.2 ECM蛋白質對於細胞貼附生長與基因表現的影響
2.4.2.1分離純化第一型膠原蛋白……………………32
2.4.2.2 分離純化第二型膠原蛋白……………………33
2.4.2.3測定膠原蛋白濃度………………………………34
2.4.2.4 PDLLA薄膜製備…………………………………35
2.4.2.5薄膜表面吸附蛋白質……………………………35
2.4.2.6細胞培養…………………………………………35
2.4.2.7細胞seeding……………………………………35
2.4.2.8細胞數測試………………………………………35
2.4.2.9 RT-PCR…………………………………………36
2.4.2.10固定細胞及染色………………………………36
2.4.2.11統計分析方法…………………………………36



第三章 結果與討論
3.1可分解高分子對軟骨細胞貼附生長及基因表現影響
3.1.1高分子薄膜製備……………….…………………….....……37
3.1.2軟骨細胞在不同可分解高分子薄膜上的貼附表現……38
3.1.3軟骨細胞在不同可分解高分子薄膜上之繁殖表現……39
3.1.4軟骨細胞在不同可分解高分子薄膜上之基因表現……40
3.2吸附不同蛋白質之PDLLA薄膜對軟骨細胞貼附生長及基因表現影響
3.2.1軟骨細胞在吸附不同蛋白質之PDLLA薄膜上1天之細胞型態及貼附表現………………………………………...............41
3.2.2軟骨細胞在吸附不同蛋白質之PDLLA薄膜表面4天、7
天及14天之型態及繁殖……………………………….…..........43
3.2.3軟骨細胞在吸附不同蛋白質之PDLLA薄膜上之基因表現…………………………………………………………...........45

第四章 結論
4.1結論………………………………………………………………76

參考文獻……………………………………………………………….......77
圖目錄

圖3.1-1不同高分子薄膜的靜態接觸角........................50
圖3.1-2軟骨細胞在不同材料表面單位面積之細胞數………………50
圖3.1-3軟骨細胞培養於不同高分子薄膜表面7天及14天上單位面積之細胞數…………………………………………….………........51
圖3.1-4尚保留部分基因型軟骨細胞在各種高分子薄膜上培養7天及14天之第一型膠原蛋白基因表現……………….…………........52
圖3.1-5尚保留部分基因型軟骨細胞在各種高分子薄膜上培養7天及14天之第二型膠原蛋白基因表現……………….…………........52
圖3.1-6尚保留部分基因型軟骨細胞在各種高分子薄膜上培養7天及
14天之aggrecan基因表現………………………………….........53
圖3.1-7已失去基因型軟骨細胞在各種高分子薄膜上培養7天及14天之第一型膠原蛋白基因表現……………………………..........54
圖3.1-8已失去基因型軟骨細胞在各種高分子薄膜上培養7天及14天之第二型膠原蛋白基因表現................................54
圖3.1-9已失去基因型軟骨細胞在各種高分子薄膜上培養7天及14天之第二型膠原蛋白基因表現.................................55
圖3.2-1在無吸附蛋白質PDLLA薄膜上1天之細胞型態…..........56
圖3.2-2 在吸附第一型膠原白薄膜上1天之細胞型態............57
圖3.2-3 在吸附第二型膠原白薄膜上1天之細胞型態............58
圖3.2-4 在吸附聚離胺酸薄膜上1天之細胞型態................59
圖3.2-5在吸附纖維網蛋白薄膜上1天之細胞型態...............60
圖3.2-6 在TCPS上1天之細胞型態............................61
圖3.2-7軟骨細胞在吸附不同蛋白質之PDLLA薄膜表面1天單位面
積之細胞數……………………….............................62
圖3.2-8細胞在吸附不同蛋白質之PDLLA薄膜表面4、7及14天單
位面積之細胞數。.........................................63
圖3.2-9 4天之細胞型態(A)未吸附蛋白質之PDLLA薄膜
(B)吸附第一型膠原白PDLLA薄膜........64
圖3.2-10 4天之細胞型態(A) 吸附第二型膠原蛋白PDLLA薄膜
(B)吸附聚離胺酸PDLLA薄膜...........65
圖3.2-11 4天之細胞型態(A)吸附纖維網蛋白PDLLA薄膜
(B)TCPS............................66
圖3.2-12 7天之細胞型態(A)未吸附蛋白質之PDLLA薄膜
(B)吸附第一型膠原白PDLLA薄膜……...67
圖3.2-13 7天之細胞型態(A) 吸附第二型膠原蛋白PDLLA薄膜
(B)吸附聚離胺酸PDLLA薄膜...........68
圖3.2-14 4天之細胞型態(A)吸附纖維網蛋白PDLLA薄膜
(B)TCPS............................69
圖3.2-15 14天之細胞型態(A)未吸附蛋白質之PDLLA薄膜
(B)吸附第一型膠原白PDLLA薄膜……..70
圖3.2-16 14天之細胞型態(A) 吸附第二型膠原蛋白PDLLA薄膜
(B)吸附聚離胺酸PDLLA薄膜..........71
圖3.2-17 14天之細胞型態(A)吸附纖維網蛋白PDLLA薄膜
(B)TCPS...........................72
圖3.2-18細胞在吸附不同蛋白質之PDLLA薄膜表面4、7及14天之第一型膠原蛋白基因表現.......................................73
圖3.2-19細胞在吸附不同蛋白質之PDLLA薄膜表面4、7及14天之第二型膠原蛋白基因表現.......................................74
圖3.2-20細胞在吸附不同蛋白質之PDLLA薄膜表面4、7及14天之aggrecan基因表現.........................................75

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Turek S.L., 傅宇輝等編譯. 骨科學原理及應用.大中國圖書公司, pp. 13-281 民76