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研究生:賴建允
研究生(外文):Jian-Yun Lai
論文名稱:拉曼光譜無損檢測之組織工程體外生成口腔黏膜等同物
論文名稱(外文):Nondestructive detection of the tissue-engineered ex-vivo produced oral mucosal equivalent with Raman spectroscopy
指導教授:江惠華江惠華引用關係
指導教授(外文):Huihua Kenny Chiang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:50
中文關鍵詞:口腔上皮細胞組織工程體外生成口腔黏膜等同物拉曼光譜
外文關鍵詞:oral keratinocytestissue engineeringex-vivo produced oral mucosal equivalentEVPOMERaman spectroscopy
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目前臨床針對口腔癌手術後上皮缺損過大的問題,主要以病患本身它處上皮來修補。後遺症則有供皮處不正常癒合、植皮處傷口收縮等併發症。利用病患自體口腔上皮細胞在無血清環境下增殖之組織工程技術,可提供安全且大量之組織用以缺損修補及修復。本研究使用拉曼光譜技術監測組織工程體外生成口腔黏膜等同物培養之成熟度,具有非破壞性和非接觸性的優點,對於生長中的口腔組織,能避免挖取染色和接觸汙染,了解組織生長情形。我們觀察到在Day1, Day7, Day14和Day21,屬於上皮細胞的拉曼訊號Amide I (1660cm-1)與Phenylalanine (1004cm-1)之peak面積比,隨著天數增加,兩者間的比值逐漸下降,趨勢線的相關係數R2=0.95,呈現越成熟比值越低的關係,可作為組織成熟度的監測依據。
Oral surgeons have problems to deal with the huge epithelial defects after surgical intervention. Traditionally, autografts of split-thickness skin graft and/or full-thickness skin graft are used and there would be lots of complications, such as donor site morbidity, graft contractility, etc. Tissue engineering based on the patient-self oral keratinocytes with serum-free environment was developed to provide safe and sufficient tissue for defect repair and regeneration. Using Raman spectroscopy could perform the quality control of the ex-vivo tissue construct with Nondestructive and non-contact detection. This study surveyed the ex-vivo produced tissue construct with non-invasive Raman spectroscopy. For the growth of oral tissue, it avoided staining and exposured to pollution which could be able to monitor the quality of the growth of maturity. We observed that in Day1, Day7, Day14 and Day21, the peak area ratio between Amide I (1660cm-1) and Phenylalanine (1004cm-1), with the number of days increased, the ratio gradually declined. The correlation coefficient was R2 = 0.95. The lower the ratio the more mature the relationship between the tissues could be used as the basis of maturity monitoring.
目錄
第一章 序論 1
1.1 研究背景 1
1.2 光學檢測方法及其特色 3
1.3 口腔黏膜與EVPOME 4
1.4 文獻回顧 7
第二章 拉曼理論 9
2.1 拉曼散射 9
2.1.1 拉曼散射演進 9
2.1.2 拉曼散射原理 10
第三章 材料與方法 16
3.1 儀器簡介 16
3.1.1 拉曼散射光譜儀 16
3.1.2 冷凍超薄切片機 19
3.2 口腔黏膜上皮 20
3.2.1 實驗流程圖 20
3.2.2 EVPOME口腔上皮培養 21
3.2.3 人體口腔上皮切片處理 24
3.3 資料分析 25
3.3.1 宇宙射線移除 25
3.3.2 減除背景螢光 25
3.3.3 峰面積積分比(peak area ratio) 27
第四章 結果與討論 28
4.1 人體口腔黏膜上皮切片分析 28
4.1.1 口腔上皮的特徵拉曼光譜圖表 28
4.1.2 口腔上皮層拉曼測量 29
4.1.3 拉曼鋒面積積分比之變化趨勢 32
4.2 EVPOME口腔上皮量測 34
4.2.1 EVPOME各天數拉曼測量 34
4.2.2 成熟天數與拉曼峰面積積分比關係 37
第五章 結論與未來展望 39
第六章 參考文獻 40



圖目錄
圖 1[5]口腔組織黏膜結構 5
圖 2體外生成口腔黏膜等同物示意圖 6
圖 3口腔組織在正常(a)、發炎(b)、癌前(c)與惡性(d)等四種光譜表徵 7
圖 4 典型的細胞光譜圖 8
圖 5散射示意圖 11
圖 6 拉曼光譜能階 15
圖 7 LabRAM HR800 拉曼光譜儀 16
圖 8拉曼光譜儀系統架構 17
圖 9 XYZ電動軸掃描平台外觀 18
圖 10 Leica CM3050 S冷凍切片機 19
圖 11整體實驗的流程圖架構 20
圖 12 零代上皮細胞培養 21
圖 13 EVPOME完全成熟照片 23
圖 14 樣本放入紙盒灌入OCT冷卻固定 24
圖 15 Leica blades- type 818 切片機專用刀片 24
圖 16 拉曼光譜處理步驟 26
圖 17 [21] 28
圖 18 各片的平均拉曼光譜 29
圖 19各片的平均拉曼減除背景螢光光譜 30
圖 20 各天數EVPOME平均拉曼光譜 34
圖 21各天數EVPOME平均拉曼減去背景光譜 35
圖 22 Day1-Day21 EVPOME染色剖面圖 36
圖 23 EVPOME成熟拉曼光譜趨勢線 38
表目錄
表格 1 32
表格 2 37
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