皮膚是對抗外來病原的第一道防線，若損傷則非常容易受到感染，因此促進傷口癒合的研究受到高度重視，也有多種促進傷口癒合材料在市場販售。利用組織工程及細胞治療已被用於促進傷口癒合，並且顯示較傳統敷料更佳的效果。本研究建立含有脂肪幹細胞類球體的膠原蛋白凝膠敷料，並測試是否能幫助傷口更快速的癒合。首先觀察脂肪幹細胞與小腸上皮細胞株C2BBe1共培養的結果，發現脂肪幹細胞能形成似微血管的管狀網絡結構。而後我們利用C2BBe1細胞層刮除一道無細胞區域，並使用不同處理模擬傷口癒合，結果顯示加入培養過脂肪幹細胞的條件培液能促使傷口癒合。接著以懸滴培養法製作脂肪細胞類球體，發現經三維培養細胞生長速率提升，並且其條件培養液能降低巨噬細胞發炎因子IL-1β的分泌。後續透過裸鼠皮膚層切除之傷口癒合模式，製造直徑為8 mm的圓形傷口，再以吻合傷口大小的含脂肪幹細胞類球體的膠原蛋白凝膠敷料覆蓋於傷口處，並與其他組比對，發現處理組的傷口具有較佳的皮膚癒合速率。組織切片結果顯示發炎性細胞的浸潤降低，傷口癒合過程中發炎時期也縮短。綜合本研究結果，此脂肪幹細胞類球體的膠原蛋白凝膠敷料對於皮膚傷口是有助益的。製備該敷料簡易，使用也便利，是促進傷口癒合的另一替代方案。

Skin is the first line of barrier to protect the body from pathogens. Patients suffering from burns, diabetes wounds and chronic skin wounds are susceptible to infections. Tissue engineering- based cell therapies have been used to promote wound healing and demonstrated to be more effective than conventional wound dressing. In this study, we examined whether adipose-derived stem cell (ADSC) spheroids, a simple three-dimensional (3D) substitute, can accelerate wound healing. First, we found ADSC co-cultured with the human intestinal epithelial C2BBe1 cells formed tube-like structure. We then investigated whether ADSC could promote cell migration using the scratch wound healing assay on monolayers of C2BBe1 cells. The result showed that wound healing was significantly enhanced after treatment with conditioned medium from ADSC. In addition, we found that ADSC-spheroids formation by hanging drop had higher cells growth rate, and ADSC conditioned medium reduced IL-1β level during inflammation. This study was followed by using the cutaneous excisional nude mice model. We covered the 8 mm-diameter wound on mice with a dressing consisting of ADSC-spheroids embedded in a collagen gel. The ADSC- spheroids collagen dressing treated-group displayed a significant faster healing rate after one week than its untreated counterpart. Data from histology sections of the wounds suggest that the quicker healing rates by ADSC-spheroids embedded collagen dressing treatment may be due to the reduction of inflammation in the wound healing process. The establishment of the ADSC-spheroids collagen dressing is beneficial to patients with skin wounds. The dressing is also simple to prepare and is convenient to be used in patients. Therefore, ADSC-spheroids collagen dressing may provide a clinical alternative to promote wound healing.

中文摘要..............................................I
Abstract.............................................II
誌謝..................................................III
縮寫字對照表...........................................IV
目錄..................................................VI
表目錄................................................VIII
圖目錄................................................IX
壹、前言..............................................1
2.1細胞培養...........................................7
2.1.1培養條件.........................................7
2.1.2小腸表皮細胞株C2BBe1繼代培養.......................7
2.1.3人類脂肪幹細胞ADSC 繼代培養........................7
2.1.4小鼠巨噬細胞株RAW264.7 繼代培養....................8
2.2管狀結構形成 (Tube-formation).......................8
2.3免疫螢光染色........................................8
2.4體外傷口癒合模式.....................................9
2.5脂肪幹細胞類球體製作與培養............................9
2.6細胞存活螢光檢測.....................................10
2.7細胞增生生長曲線.....................................10
2.8巨噬細胞RAW264.7發炎反應試驗..........................10
2.9酵素結合免疫吸附分析 (ELISA) 檢測發炎因子表現量.........11
2.10三維膠原蛋白凝膠製備 (3D collagen gel)...............11
2.11裸鼠皮膚層傷口癒合模式...............................12
2.12免疫組織化學染色....................................12
2.13免疫組織螢光染色....................................12
2.14細胞層片製作 (Cell sheet)...........................13
2.15表皮化脂肪幹細胞之分化...............................14
2.16數據統計分析........................................14
參、結果................................................15
3.1脂肪幹細胞ADSC與小腸上皮細胞株C2BBe1共培養的形態........15
3.2管狀結構形成對脂肪幹細胞特性的影響......................15
3.3脂肪幹細胞條件培養液對小腸上皮細胞株移動能力的影響........16
3.4脂肪幹細胞類球體對生長速率的影響........................17
3.5脂肪幹細胞類球體條件培養液對發炎反應的影響...............17
3.6脂肪幹細胞類球體於膠原蛋白凝膠設計與製作.................17
3.7裸鼠皮膚傷口癒合模式I..................................18
3.8裸鼠皮膚傷口癒合模式II.................................19
3.9維他命C衍生物A2-P形成細胞組織層對細胞形態的影響...........20
3.10脂肪幹細胞培養於嵌入式培養皿與分化培養液對細胞形態的影響..20
肆、討論.................................................22
伍、參考文獻.............................................27

中文摘要..............................................I
Abstract.............................................II
誌謝..................................................III
縮寫字對照表...........................................IV
目錄..................................................VI
表目錄................................................VIII
圖目錄................................................IX
壹、前言..............................................1
2.1細胞培養...........................................7
2.1.1培養條件.........................................7
2.1.2小腸表皮細胞株C2BBe1繼代培養.......................7
2.1.3人類脂肪幹細胞ADSC 繼代培養........................7
2.1.4小鼠巨噬細胞株RAW264.7 繼代培養....................8
2.2管狀結構形成 (Tube-formation).......................8
2.3免疫螢光染色........................................8
2.4體外傷口癒合模式.....................................9
2.5脂肪幹細胞類球體製作與培養............................9
2.6細胞存活螢光檢測.....................................10
2.7細胞增生生長曲線.....................................10
2.8巨噬細胞RAW264.7發炎反應試驗..........................10
2.9酵素結合免疫吸附分析 (ELISA) 檢測發炎因子表現量.........11
2.10三維膠原蛋白凝膠製備 (3D collagen gel)...............11
2.11裸鼠皮膚層傷口癒合模式...............................12
2.12免疫組織化學染色....................................12
2.13免疫組織螢光染色....................................12
2.14細胞層片製作 (Cell sheet)...........................13
2.15表皮化脂肪幹細胞之分化...............................14
2.16數據統計分析........................................14
參、結果................................................15
3.1脂肪幹細胞ADSC與小腸上皮細胞株C2BBe1共培養的形態........15
3.2管狀結構形成對脂肪幹細胞特性的影響......................15
3.3脂肪幹細胞條件培養液對小腸上皮細胞株移動能力的影響........16
3.4脂肪幹細胞類球體對生長速率的影響........................17
3.5脂肪幹細胞類球體條件培養液對發炎反應的影響...............17
3.6脂肪幹細胞類球體於膠原蛋白凝膠設計與製作.................17
3.7裸鼠皮膚傷口癒合模式I..................................18
3.8裸鼠皮膚傷口癒合模式II.................................19
3.9維他命C衍生物A2-P形成細胞組織層對細胞形態的影響...........20
3.10脂肪幹細胞培養於嵌入式培養皿與分化培養液對細胞形態的影響..20
肆、討論.................................................22
伍、參考文獻.............................................27

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