臺灣博碩士論文加值系統

大本山葡萄是具有抗發炎反應及止血能力的中藥材，利用乙醇回流萃取法進行有效成分萃取與分析，透過添加LPS刺激巨噬細胞RAW264.7發炎反應，產生IL-1β, IL-6, TNF-α等細胞激素與一氧化氮(nitric oxide，NO)生成，再加入大本山葡萄萃取物，評估其抗發炎的效果並找出最佳抗發炎的萃取物添加濃度。動物實驗測試，以奈米殼聚醣電紡絲作為吸附載體，浸泡在大本山葡萄萃取液中再覆蓋在大鼠背部傷口處，觀察傷口癒合程度，對組織分別作H&E染色及Masson’s 染色，觀察組織癒合程度及膠原蛋白沉積量，評估在傷口癒合的階段和癒合效果。經過6天敷藥後，傷口復原達91%，只覆蓋紗布的控制組只有48%癒合，加藥治療可在12天完全復原。組織切片染色中的H&E及膠原蛋白染色中， 結果都顯示添加大本山葡萄萃取液有明顯傷口癒合效果，其中又以殼聚醣奈米纖維膜當作載體最為明顯。而膠原蛋白一型的定量中，無論是第6天還是第12天來看也是添加大本山葡萄萃取液的有很多的膠原蛋白濃度，以上數據呈現大本山葡萄萃取液確實有加速傷口癒合的功效，未來可進一步確定萃取成份，應用到臨床皮膚傷口治療。

Ampelopsis brevipedunculata, a chinese herbal medicine has anti-inflammatory and hemostatic ability. In this study, we extract the curing components by the reflux extraction of ethanol and analyze the effective components. Induced the inflammation of RAW264.7 cells by LPS and expressed some cytokines such as IL-1β, IL-6, TNF-α, and produce nitric oxide (NO), and then added the extracts of Ampelopsis brevipedunculata into medium to estimate the curing effect and found out the optimal adding concertation. In animal experiment, nanofibers of chitosan soaked in the extract of Ampelopsis brevipedunculata solution, and then covered on the wound position in the back of rat. To estimate the curing effect of extract Ampelopsis brevipedunculata, we observed the wound area recovery, organizational staining of H&E and Masson and the collage deposition. There is 91% wound recovery for the extract Ampelopsis brevipedunculata adding, and only 48% wound recovery for swab after 6 days curing. The full recovery of wound at 12 days by adding the extract Ampelopsis brevipedunculata, and the results of the organizational staining and collage deposition appeared the fast curing effect. To confirm the effect compositions of Ampelopsis brevipedunculata in wound curing and can be used to the clinical curing applications in further.

目錄

中文摘要 I
ABSTRACT II
致謝 V
表目錄 X
圖目錄 XI
第一章 緒論 1
1.1 前言…...……………………………………….1
1.2 研究動機目的…………………………..…2
第二章 文獻回顧 3
2.1皮膚結構 3
2.2傷口癒合機制(Wound healing)…………….…….5
2.3免疫機制(Immune system)………………….…….7
2.3.1發炎反應……………………………….5
2.3.2發炎機制…………………………………….6
2.3.3巨噬細胞(Macrophages)………….7
2.4敷料………………………………….....9
2.4.1敷料歷史………………………………….8
2.4.2敷料種類…………………………………9
2.5奈米纖維……………………………11
2.5.1靜電紡絲…………………………9
2.5.2奈米電紡原理………………………10
2.5.3影響靜電紡絲製作因素.......10
2.5.1靜電紡絲應用及優勢………….……..11
2.6 生醫材料…………………….14
2.6.1生醫材料種類………………..11
2.6.2幾丁質……………….…..12
2.6.3幾丁聚醣………………………………..13
2.6.4細菌纖維素………………………….….14
2.7大本山葡萄……………………………..…19
2.7.1大本山葡萄介紹………………19
2.7.2大本山葡萄成分…………………19
2.8 膠原蛋白(Collagen) ………………………21
2.8.1免疫組織染色…………………….21
2.8.2 Hematoxylin eosin stain(H&E stain)….20 2.8.3 Collagen-Masson’s Trichrome Stain(TRS)…21
2.8.4 ELISA…………………………22
第三章 材料與方法 24
3.1 實驗流程 25
3.2 實驗材料 26
3.2.1 實驗儀器設備 27
3.3實驗方法…………………………………………. 34
3.3.1萃取大本山葡萄 28
3.4濃度測試...............................28
3.4.1 總多酚濃度測試.......................28
3.4.2 抗氧化分析.......................29
3.4.3萃取率………………………………………29
3.5材料製作…………………………………28
3.5.1電紡製作………………………………29
3.5.2細菌纖維素製作…………………….29
3.5.2.1培養基配置……………...29
3.5.2.2細菌纖維素培養……………………..….29
3.5.3拉伸強度.........................29
3.6細胞實驗…………………………………31
3.6.1 MTT測試……………………30
3.6.2 NO 測試……………………………...31
3.6.3 NF-κB濃度測試………………31 　　　　　　　　　　　　　　　　　　　　
　　　　　3.6.4 ROS分析(細胞內活性氧族群). ……31
3.7動物實驗(In vivo)………………………….35
3.7.1全層皮膚創口操作…………..….32
3.7.2 組織包埋及切片……… ........33
3.7.3 Haematoxylin and Eosin Stain……….33
3.7.4　Collagen- Massons Trichrome Stain..34
　　　3.7.5膠原蛋白定量(collage type I )………….35

第四章 實驗結果 34
4-1細菌纖維素及殼聚醣奈米纖維膜示意圖........38
4-2細菌纖維素及奈米纖維膜的SEM圖…………………38
4-3殼聚醣奈米纖維及細菌纖維素之拉伸強度……38
4-4殼聚醣纖維膜及細菌纖維素之降解速率……….40
4-5 不同濃度大本山葡萄萃取液對細胞之存活率……40
4-6 不同濃度大本山萃取液抑制NO濃度……………41
4-7不同濃度大本山萃取液抑制NF-κB濃度……42
4-8不同濃度大本山萃取液抑制ROS濃度…………43
4-9 傷口癒合示意圖……………………………….45
4-10:傷口癒合速率( Wound healing(%) )……46
4-11:傷口癒合面積(Residual Wound Area(mm2)....46
4-12為第六天不同組別之伊紅染色(H&E)…………….47
4-13為第六天不同組別之三色染色(Masson’s stain)…48
4-14為第十二天不同組別的伊紅染色(H&E)………49
4-15為第十二天不同組別之三色染色(Masson’s stain)....50
4-16第六天膠原蛋白一型定量………………51
4-17第十二天膠原蛋白一型定量……………52
4-18 氣相色譜法(GC-MS)定性分析………….54



第五章 實驗討論 56
參考文獻 62

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