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論文中文名稱:低頻率電磁場對於貼附在幾丁聚醣薄膜上之軟骨細胞之影響 [以論文名稱查詢館藏系統]
論文英文名稱:The effect of EMF stimulation to chondrocyte that planted on chitosan film [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:97
出版年度:98
中文姓名:蕭翊瑋
英文姓名:Yi-Wei Hsiao
研究生學號:95688015
學位類別:碩士
語文別:中文
口試日期:2009-05-27
論文頁數:68
指導教授中文名:林忻怡
指導教授英文名:Hsin-Yi Lin
口試委員中文名:黃聲東;蔡偉博
口試委員英文名:Sheng-Tung Huang;Wei-Bor Tsai
中文關鍵詞:幾丁聚醣薄膜低頻率電磁波軟骨細胞
英文關鍵詞:chitosanlow-frequency pulsed electromagneticchondrocyte
論文中文摘要:低頻率電磁場目前在臨床使用上,能用來治療骨關節炎使病人之患部減輕發炎反應,以減低骨關節炎對於關節軟骨之損壞。有研究指出電磁場可以增進受損軟骨組織的修復,然而當體內軟骨組織缺損過大時,電磁場之治療無法有明顯之效果,因此須藉由導入載有細胞之組織工程材料來修補缺損。以幾丁聚醣所製作織細胞支架,具有細胞相容性、無毒性以及可分解之特性,因此具有刺激軟骨生長及痊癒後不需再經手術取出之優點。
本實驗研究目的為測試以幾丁聚醣為支架來修復軟骨組織的情況下,若再以電磁場照射,是否能產生有如臨床之修復效果。
實驗中將軟骨細胞培養在去乙醯基化程度96%之幾丁聚醣所製成之薄膜,並以每天2小時電磁場(電磁場參數:強度:18-33 G、頻率:75 Hz、脈衝寬:1.3 ms、振幅:3.5±1 mV)照射,照射時間達21天,並於第7、14、21天來測試軟骨細胞之反應。實驗主要測定項目為細胞活性(MTT assay)、細胞增殖狀況(dsDNA content)以及軟骨細胞特定胞外基質(GAG 及type II collagen content)之染色與分泌量。對照組為未經電磁場照射之細胞。從實驗結果得知軟骨細胞在照射完電磁波後其細胞活性及細胞增殖量和對照組相比雖有些微增加但無明顯差異;兩組之葡萄醣胺聚醣及第二型膠原蛋白染色結果均證實有正常分泌之情況,但因以肉眼觀察無法辨別明顯差異故藉由葡萄醣胺聚醣定量法(DMMB assay)以及第二型膠原蛋白免疫定量法(Type II collagen-ELISA assay)來定量探討實驗組與對照組胞外基質之分泌。由實驗結果得知實驗組之葡萄醣胺聚醣分泌量在21天,有明顯多於控制組;而實驗組之第二型膠原蛋白之分泌量於第14及21天,有明顯多於控制組。
由本實驗之結果推測電磁場對於貼附於幾丁聚醣薄膜之豬軟骨細胞之細胞活性、細胞增殖雖有增加但並無統計學上之差異,然而對於胞外基質葡萄醣胺聚醣及第二型膠原蛋白之分泌量有明顯改善之情況。
論文英文摘要:Low Frequency Pulsed Electromagnetic Field (EMF) has been used in clinical application to treat osteoarthritis (OA). OA patients receiving EMF treatment have been shown to have reduced pain and inflammatory reaction. Literatures have also shown that EMF can improve articular cartilage repair. However, the healing effect on EMF on lesion larger than 1cm is unsatisfactory. Biocompatible tissue engineered (TE) constructs seeded with chondrocytes or mesenchymal stem cells are usually used to fill larger lesions and to accelerate the repair process. Among the biocompatible materials, chitosan resembles extracellular matrix of cartilage. It is biodegradable and easily available from nature. Chitosan TE scaffolds have been shown to enhance chondrocyte growth and extracellular matrix (ECM) deposition.
This experiment exposed chondrocyte cells cultured on chitosan films to EMF to simulate in vitro tissue engineering condition. Tests were done on cells to assay whether EMF could reproduce its positive in vivo healing effects in an in vitro tissue engineering model.
Primary porcine chondrocytes were seeded on chitosan (96% deacetylated) films and exposed to EMF (19-33 Gauss, 75Hz, duty cycle 1.3ms) two hours a day for 21 days. The cells were tested on day 7, 14, and 21 for their viability, proliferation, type II collagen and glycosaminoglycan (GAG) release. The cells were also stained for GAG and type II collagen and prepared for SEM imaging.
The results showed EMF cell proliferation and viability were similar to those of controls in weeks 1, 2, 3. Both groups had normal GAG and type II collagen release according to results from immunohistological staining. The amount of GAG release by the EMF cells was significantly higher than that released by controls in week 3. Also the amount of type II collagen produced by EMF cells was higher than that by controls in weeks 2 and 3.
Based on the results from this experimental setup, we found EMF could benefit articular cartilage repair when tissue engineered scaffolds are used. Though EMF simulated did not seem to stimulate cell growth, it accelerated cells’ ECM deposition.
論文目次:目 錄

中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
表目錄 ix
圖目錄 x
第一章 緒論 1
1.1 關節軟骨相關之介紹 1
1.1.1 關節軟骨結構及組成 1
1.1.2 關節軟骨損傷及治療方法簡介 5
1.2 幾丁聚醣之相關簡介 6
1.2.1 幾丁質之起源及幾丁質之衍生物-幾丁聚醣之簡介 6
1.2.2 幾丁聚醣之特性及組織工程相關研究 8
1.3 電磁場相關簡介 10
1.3.1 電磁場簡介 10
1.3.2 電磁場用於醫療研究之簡介 11
1.3.3 低頻率電磁場於組織工程之相關研究 12
1.4 研究目的 15
第二章 實驗儀器、材料、步驟 16
2.1 實驗儀器 16
2.2 實驗藥品 17
2.2.1 製作幾丁聚醣薄膜所需藥品 17
2.2.2 取豬軟骨細胞及培養軟骨細胞所需藥品 18
2.2.3 實驗測定所需藥品 19
2.3 實驗方法 21
2.3.1 電磁場產生器 21
2.3.2 幾丁聚醣薄膜製作 22
2.3.2.1 10X磷酸緩衝溶液製備方法 23
2.3.3 取得初代豬軟骨細胞之方法與細胞培養 24
2.3.3.1 取得初代豬軟骨細胞之方法 24
2.3.3.2 初代豬軟骨細胞培養 25
2.3.4 軟骨細胞之活性測試及染色 27
2.3.4.1 細胞活性(MTT assay) 27
2.3.4.2 DNA含量測定 28
2.3.4.3 葡萄醣胺聚醣染色(GAG stain) 28
2.3.4.4 第二型膠原蛋白免疫染色 29
2.3.4.5 葡萄醣胺聚醣含量測定 29
2.3.4.6 第二型膠原蛋白含量測定 30
2.3.4.7 電子式顯微鏡拍照(SEM) 31
2.3.4.8 統計分析 32
第三章 實驗結果 33
3.1 軟骨細胞於幾丁聚醣薄膜上之活性測試結果 33
3.1.1 DNA含量 33
3.1.2 細胞活性(MTT assay) 34
3.1.3 葡萄醣胺聚醣染色(Glycosaminoglycan stain) 35
3.1.4 第二型膠原蛋白染色(Type II collagen stain) 36
3.1.5 葡萄醣胺聚醣含量(Glycosaminoglycan content) 37
3.1.6 第二型膠原蛋白含量(Type II collagen content) 38
3.1.7 電子式顯微鏡(SEM) 39
第四章 結果與討論 42
參考文獻 45
附錄
1. DNA含量標準曲線………………………………………………...52
2. chondroitin-6-sulfate 含量標準曲線………………………………...53
3. Type II collagen 含量標準曲線…………………… ……………….54
4.實驗條件設定進程………………………………………………...…55
5.人類軟骨細胞培養於幾丁醣支架並照射電磁場實驗……………56
6.人類軟骨細胞分別培養於細胞培養盤及幾丁醣支架實驗….…...59
7.人類軟骨細胞培養於細胞培養盤並照射電磁場實驗……………62
8.初代豬軟骨細胞培養於幾丁聚醣薄膜並照射電磁場實驗….…...66
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