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系統識別號 U0026-0812200910461153
論文名稱(中文) 鹼性纖維母細胞生長因子於膠原蛋白-透明質酸基質中對於傷口癒合之作用
論文名稱(英文) The effects of bFGF in collagen-hyaluronan matrix on wound healing
校院名稱 成功大學
系所名稱(中) 生物科技研究所
系所名稱(英) Institute of Biotechnology
學年度 91
學期 2
出版年 92
研究生(中文) 林獻珍
研究生(英文) Hsien-Chen Lin
電子信箱 kulocon@yahoo.com.tw
學號 l6690112
學位類別 碩士
語文別 中文
論文頁數 68頁
口試委員 口試委員-王盈錦
指導教授-黃玲惠
口試委員-戴明泓
口試委員-胡孝光
口試委員-謝式洲
中文關鍵字 血管新生  鹼性纖維母細胞生長因子  傷口癒合  基質 
英文關鍵字 bFGF  PCM-AH  matrix  wound healing  Integra  angiogenesis 
學科別分類
中文摘要 鹼性纖維母細胞生長因子(basic Fibroblast Growth Factor;bFGF)在傷口癒合的應用上已證實有促進作用,其中包含:促進早期的粒狀組織(granulation tissue)生成、葡萄胺聚醣(glycosaminoglycan;GAG)沉積、Fibronectin生成、纖維母細胞增生、較佳的膠原蛋白交聯、減少疤痕形成、增加癒合皮膚張力、促進血管新生(angiogenesis)、促進發炎細胞滲透等作用。加上本實驗室所研發之基質(PCM-AH)已經動物實驗證實有促進傷口癒合的效果,因此本研究之主要目的是進一步將bFGF添加於PCM-AH基質中,用來覆蓋在2x3 cm2之全深度(full-thickness)傷口,並以人工皮膚Integra®作為對照,觀察各種基質對傷口癒合的影響。
動物實驗是以天竺鼠(Hartley品系)為模式動物,經麻醉、剃毛、消毒後,將在天竺鼠背部開四個2x3 cm2的全深度傷口,每個傷口個別覆蓋上一種基質,分別是第一組PCM-AH-bFGF(40μg)、第二組bFGF(40μg)、第三組PCM-AH、第四組Integra®、第五組open control;觀察的時間選擇在手術後的第6、12、24、120天犧牲動物,並取下背部皮膚進行包埋、切片與染色分析。觀察的結果發現:PCM-AH-bFGF組在外觀閉合速度上接近正常癒合的速度,然而Integra®組卻有延遲癒合的現象;在癒合初期時基質中細胞增生的觀察發現PCM-AH-bFGF、bFGF兩組有吸引發炎細胞移入的現象,第6天的纖維母細胞密度是PCM-AH-bFGF、bFGF與PCM-AH三組較高,但第12天時bFGF與open control兩組的纖維母細胞密度卻高出其他組別,比起第6天的結果,兩者約莫增加了兩倍的細胞數,此一結果很可能與傷口收縮有關;在血管新生方面,PCM-AH與bFGF兩組個別有促進血管新生的作用,當PCM-AH與bFGF共同處理傷口時,血管新生的有加成的現象;新生皮膚厚度定量方面,PCM-AH-bFGF、bFGF、PCM-AH等3組在促進纖維增生上皆比open control與Integra®兩組要來的顯著,但無法從數據中看出PCM-AH-bFGF組在這方面有何差異;上皮厚度的觀察上各組皆有變厚的現象,到120天仍未有完全恢復到正常厚度的顯著趨勢,故在這方面或許要更長的時間才能觀察到;在癒合組織型態、膠原蛋白走向以及彈性纖維表現的觀察上,PCM-AH-bFGF組皆有較接近正常皮膚組織的結果,其次PCM-AH與bFGF兩組也都有較接近正常組織的現象。由一系列的動物實驗觀察結果顯示在PCM-AH基質添加bFGF對於傷口癒合有良好的促進效果,在臨床上具有應用價值與潛力。
英文摘要 The promoted functions on wound healing application of basic fibroblast growth factor(bFGF)had been confirmed. These functions include that the promotion of the growth of granulation tissue in early stage, accumulation of glycosaminoglycan(GAG), production of fibronectin, proliferation of fibroblast, cross linking of collagen, increase the tensile strength of healing skin, promotion of angiogenesis, infiltration of inflammatory cells, and reduce the scar formation etc. Excellent collagen matrices called “PCM-AH” which is suitable for skin tissue engineering has developing in our lab. And it was evident that these matrices could promote wound healing. Therefore, the main purpose of this study is to combine bFGF with PCM-AH and to investigate its effect on wound healing.

Briefly, four 2x3 cm2 full-thickness wounds will be created dorsally on adult male Hartley Guinea pigs. The effects of PCM-AH with bFGF(40μg)will be compared with PCM-AH only, bFGF(40μg)only, open control and Integra, a commercialized artificial skin, on wound healing process. The time interval of animal sacrificed is planned on days 6, 12, 24 and 120 after surgery. The dorsal skin will be take and embed in resin. Many observations will carry out by plastic section and some staining methods. The results of these experiments can be found that include:The healing rate of PCM-AH with bFGF group was more close to normal healing rate but Integra group delay the wound healing. The PCM-AH with bFGF group and bFGF only group can attract inflammatory cell to migrate in matrices in the observations of cell proliferation in matrices in primary healing staged on day 6. The fibroblast density in matrices of PCM-AH-bFGF group、bFGF group and PCM-AH group are higher than other two group on day 6. But bFGF group and open control group are higher than others on day 12. This result compare with day 6 will be find both them increase around two-fold cell number. It can be supposed the cell density is in connection with wound contraction. In the angiogenesis study, both PCM-AH and bFGF can promote angiogenesis. When PCM-AH and bFGF use together to treat the same wound, the angiogenesis phenomenon wound be increased. In the quantitative experiments of regeneration skin thickness, the PCM-AH-bFGF group, bFGF group and PCM-AH group promote the fibroplasias than other two groups. But it can’t be found the PCM-AH-bFGF group to have any difference in this data. In the observation of epidermal thickness, all of them are thicker than normal epidermis. Until day 120, they are not to restore to normal. So it may be needed to observe more long time. In the healing tissue morphology study, collagen arrangement orientation study and elastin expression study, the PCM-AH-bFGF group is more close to normal tissue. The PCM-AH group and bFGF group are secondary. Due to a serious of animal experiment experiments, it is reveal that the PCM-AH matrix with bFGF can promote good wound healing. This combination is very valuable and potential on clinical exercise.
論文目次 目錄
中文摘要
英文摘要
誌謝
目錄
表目錄
圖目錄
符號
第一章 緒論…………………………………………………….… 1
1.1 傷口癒合…………………………………………………… 1
1.1.1 傷口癒合機制……………………………………………… 2
1.1.2 不正常傷口癒合…………………………………………… 4
1.1.3 目前對於傷口癒合的評估方式…………………………… 4
1.2 組織模板…………………………………………………… 5
1.2.1 組織模板的應用性與重要性……………………………… 5
1.2.2 目前以商品化的組織模板………………………………… 6
1.2.3 本次實驗用的基質………………………………………… 6
1.3 纖維母細胞生長因子……………………………………… 6
1.3.1 目前對於鹼性纖維母細胞生長因子之研究……………… 7
1.3.2 鹼性纖維母細胞生長因子對於傷口癒合之影響………… 7
第二章 實驗藥品、儀器與方法…………………………………. 23
2.1 實驗藥品…………………………………………………… 23
2.2 實驗儀器設備……………………………………………… 25
2.3 實驗方法…………………………………………………… 27
2.3.1 多孔性膠原蛋白基質移植入天竺鼠全深度傷口之手術… 27
2.3.2 天竺鼠新生皮膚組織之固定,脫水,包埋,切片………. 28
2.3.3 Hematoxylin and eosin染色(H&E染色)………………. 29
2.3.4 Masson氏三色(trichrome)染色………………………… 29
2.3.5 Verhoeff氏彈性纖維染色…………………………………. 30
第三章 鹼性纖維母細胞生長因子於膠原蛋白透明質酸基質中對於傷口癒合之影響 32
3.1 實驗目的…………………………………………………… 32
3.2 實驗材料與方法…………………………………………… 32
3.2.1 傷口表面癒合情形………………………………………… 32
3.2.2 傷口癒合初期,基質中之細胞增生情形………………..… 32
3.2.3 傷口癒合初期的血管新生情形…………………………… 33
3.2.4 新生皮膚厚度之量測……………………………………… 33
3.2.5 新生皮膚上皮新生之比較………………………………… 34
3.2.6 傷口內部癒合情形………………………………………… 34
3.2.7 膠原蛋白走向……………………………………………… 34
3.2.8 癒合後彈性纖維表現……………………………………… 35
3.3 實驗結果…………………………………………………… 35
3.3.1 傷口表面癒合情形………………………………………… 35
3.3.2 傷口癒合初期,基質中細胞增生情形……………..……… 35
3.3.3 傷口癒合初期的血管新生情形…………………………… 36
3.3.4 新生皮膚厚度之量測……………………………………… 36
3.3.5 新生皮膚上皮新生之比較………………………………… 36
3.3.6 傷口內部癒合情形………………………………………… 37
3.3.7 膠原蛋白走向……………………………………………… 37
3.3.8 癒合後彈性纖維表現……………………………………… 37
3.4 實驗討論…………………………………………………… 38
第四章 結論……………………………………………………... 60
第五章 參考文獻………………………………………………... 62
表目錄
表 1.1 參與傷口癒合過程的細胞激素與生長因子及其作用….. 9
表 1.2 Integrin family及其ligands………………………………. 10
表 1.3 目前觀察傷口癒合的研究方法………………………….. 11
表 1.4 目前已商品化之皮膚替代物…………………………….. 21
表 3.1 傷口癒合初期,基質中細胞增生之比較………………… 45
表 3.2 第十二天傷口血管新生之比較………………………….. 47
圖目錄
圖 1.1 傷口癒合的過程………………………………………….. 8
圖 3.1 傷口表面癒合情形………………………………………... 41
圖 3.2 傷口癒合初期,基質中之細胞增生情形…………………. 42
圖 3.3 第12天傷口血管新生情形……………………………… 46
圖 3.4 第12天傷口血管新生之比較…………………………… 47
圖 3.5 第12、24、120天之新生皮膚厚度變化……………….. 48
圖 3.6 第12、24、120天之新生皮膚厚度定量結果………….. 49
圖 3.7 第12、24天上皮新生,上皮移入傷口之距離….............. 50
圖 3.8 第12、24、120天新生表皮厚度定量……………………. 51
圖 3.9 第24、120天癒合組織型態……………………………. 52
圖 3.10 第24、120天癒合組織的膠原蛋白分佈情形………….. 54
圖 3.11 第120天癒合組織中彈性纖維表現情形……………….. 56
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