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研究生:林郁敏
研究生(外文):Yu-Min Lin
論文名稱:引導骨組織再生膜之細胞模式研究
論文名稱(外文):In Vitro Study of Guided Bone Tissue Regeneration Membrane
指導教授:鍾仁傑鍾仁傑引用關係
口試委員:曾靖孋蔡偉博林忻怡
口試日期:2012-07-09
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:82
中文關鍵詞:第一型膠原蛋白低聚合原花青素引導組織再生膜細胞實驗
外文關鍵詞:Type I CollagenOligomeric ProanthocyanidinsGuided Tissue Regeneration MembraneIn Vitro Study
相關次數:
  • 被引用被引用:1
  • 點閱點閱:238
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
本研究旨在進行引導骨組織再生膜之細胞模式研究。以原花青素與第一型膠原蛋白進行交聯反應,並製備出含 10 % 原花青素之組織再生薄膜 (簡稱為P-C film),實驗以人類類骨母細胞 (MG63) 與人類皮膚纖維母細胞 (WS1) 細胞株進行研究。評估類骨母細胞對 P-C film 之細胞毒性、貼附、增生、分化的影響。並利用孔徑分析儀分析材料孔徑率,以及將 MG63 與 WS1 進行共培養實驗來探討組織再生膜是否能阻止細胞移行穿透,並達到阻隔的作用。
研究結果顯示,在 MTT assay 與細胞週期細胞活性測試方面,P-C film 不影響 MG63 類骨母細胞之生長。在 SEM 及 H33258 螢光染色細胞貼附測試方面,隨著 P-C film 與 MG63 cell 貼附的時間增長,細胞貼附於組織再生膜上之數目隨之增加。在細胞增生與分化測試方面,P-C film 能刺激 MG63 cell 的增生,提早骨母細胞於第 7 天開始進行骨分化,並於第 14 天觀察到鈣沉積的生成。而於組織再生膜之細胞穿透性探討方面,P-C film 孔洞分佈範圍在 3.3 ~ 101.6 μm,其中孔洞平均直徑範圍落在 7 ~ 9 μm,得知組織再生膜孔徑非常小。於細胞穿透性測試方面,經由 MG63 cell 與 WS1 cell 單面培養,細胞貼附於材料表面情形良好,且材料下方並無細胞核被染出,可知我們的組織再生膜確實具有阻隔細胞穿透之功能。
綜合以上結果,此組織再生膜具有良好的生物相容性,不僅可刺激骨母細胞的增生及分化,還具有阻隔細胞移行穿透的功能,故此研究製備的 P-C film 在引導骨組織再生膜的應用上有很大的潛力。


The purpose of this research was to carry out the detailed in vitro study of guided bone tissue regeneration membrane. The fabricated membrane, P-C film, was made of type I collagen cross-linked using oligomeric proanthocyanidins (OPCs) as the cross-linking agent. The human osteoblast cell line (MG63) and human skin fibroblast cell line (WS1) were cultured for in vitro studies. The effects of P-C film on MG63 cell viability, adhesion, proliferation, alkaline phosphatase activity and differentiation were evaluated. In order to investigate the barrier ability of the tissue regeneration membrane, capillary flow porometer was used to analyze the pore size; separated co-culture experiments using MG63 and WS1 were carried out to observe the cell migration behavior.
The results showed that P-C film did not affect cell growth and had good biocompatibility. The cells attached to the P-C film surface increased with time. We also found that MG63 cells grew faster on P-C film, compared to the culture flask. On the other hand, P-C film promoted osteoblast to enter the differentiation stage, indicated by higher alkaline phosphatase activity and calcium deposition. In addition, the mean pore size of P-C film ranged at 7~9 μm, which was small enough to block the migration of cells through the membrane.
In conclusions, P-C film does not affect cell growth, and is able to promote the differentiation of osteoblast. Results of in vitro experiments indicate that P-C film has great potential in the applications of guided bone regeneration and bone tissue engineering.


中文摘要 I
ABSTRACT III
致謝 V
目錄 VI
圖目錄 X
表目錄 XIII
第一章 緒論 1
1-1、前言 1
1-2、研究動機與目的 3
1-3、研究流程與章節說明 3
第二章 文獻回顧 4
2-1、引導組織再生膜 4
2-1-1、引導組織再生膜介紹 4
2-1-2、引導組織再生膜之臨床上的應用 4
2-2、骨組織 10
2-2-1、骨之結構 10
2-2-2、骨細胞組成 11
2-2-3、骨成型模型之膜內骨化 12
2-2-4、骨分化標誌 14
2-3、膠原蛋白 16
2-4、低聚合原花青素 18
2-4-1、低聚合原花青素的介紹 18
2-4-2、低聚合原花青素的交聯鍵結反應 20
2-5、生物相容性 22
2-5-1、生物相容性概述 22
2-5-2、生物相容性評估 23
第三章 實驗材料與方法 25
3-1、實驗藥品與儀器 25
3-1-1、藥品與耗材 25
3-1-2、儀器設備 30
3-1-2.1、酵素連結免疫吸附分析儀 (ELISA Reader) 30
3-1-2.2、流式細胞儀 (Flow Cytometer) 31
3-1-2.3、掃描式電子顯微鏡 (Scanning Electron Microscope, SEM) 32
3-1-2.4、倒立式光學/螢光顯微鏡 33
3-1-2.5、紫外光/可見光分光光譜儀 (Ultraviolet/Visible Spectrophotometer, UV-Visible) 34
3-1-2.6、毛細流孔徑分析儀 (Capillary Flow Porometer) 35
3-1-3、其他儀器 36
3-2、實驗流程 37
3-3、材料製備 38
3-3-1、膠原蛋白去免疫端 38
3-3-2、組織再生膜之製備 38
3-4、體外細胞膜式實驗流程 41
3-5、體外細胞模式實驗 44
3-5-1、細胞繼代培養 44
3-5-2、組織再生膜對 MG63 類骨母細胞活性測試 44
3-5-2.1、MTT assay 44
3-5-2.2、細胞週期檢測 45
3-5-3、MG63類骨母細胞貼附於組織再生膜之表面形貌觀察 46
3-5-3.1、掃描式電子顯微鏡觀察 46
3-5-3.1、螢光顯微鏡觀察 47
3-5-4、MG63類骨母細胞培養於組織再生膜之細胞增生測試 48
3-5-4.1、DNA 含量檢測 48
3-5-5、MG63類骨母細胞培養於組織再生膜之細胞分化測試 49
3-5-5.1、ALP 活性分析 49
3-5-5.2、Von Kossa 鈣沉積染色分析 50
3-5-6、組織再生膜之細胞穿透性分析 50
3-5-6.1、毛細流孔徑分析 50
3-5-6.2、細胞穿透分析 51
第四章 結果與討論 53
4-1、組織再生膜對 MG63 類骨母細胞細胞活性檢測分析結果 53
4-1-1、MTT assay 檢測分析結果 53
4-1-2、細胞週期檢測分析結果 54
4-2、MG63類骨母細胞貼附於組織再生膜之表面形貌觀察分析結果 58
4-2-1、掃描式電子顯微鏡觀察分析結果 58
4-2-2、螢光顯微鏡觀察分析結果 59
4-3、組織再生膜對 MG63 類骨母細胞細胞增生分析結果 61
4-3-1、DNA 含量檢測分析結果 61
4-4、MG63類骨母細胞培養於組織再生膜之細胞分化分析結果 63
4-4-1、ALP 活性檢測分析結果 63
4-4-2、Von Kossa 檢測分析結果 66
4-5、組織再生膜之細胞穿透性檢測分析結果 68
4-5-1、孔徑分析儀檢測分析結果 68
4-5-2、細胞穿透分析結果 69
4-5-2.1、單面培養分析 69
第五章 結論 73
參考文獻 75




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