禿髮在現今社會普遍的問題，影響到越來越多年齡層的男女，因為直接影響外觀，對於患者的心理負擔更是嚴重。目前禿髮的標準治療使用像是藥物治療、光治療或植髮等，然而這些治療的成本都較高，但是卻有治療效果不穩定性及藥物副作用等可能的問題。近期隨著組織工程的發展與趨勢有越來越多學者、團隊使用組織工程的方式來進行毛囊再生之研究。毛囊再生研究與屬於特化間葉組織的真皮乳頭細胞息息相關，但真皮乳頭細胞會隨著繼代培養次數上升而降低其誘導毛髮生長的能力。毛髮發育是透過表皮—間葉之間的相互作用產生，若能使毛囊細胞與表皮細胞共同培養聚集形成立體球狀構造，以促進彼此的交互作用，使毛囊保持其毛髮誘導的能力。

因此本研究的目的在於提供一種簡易的方法用來製備及培養真皮乳頭細胞和表皮細胞形成的微球組織，有可以達到高產量、大小均一的特點，且觀察其是否能在生物體內的環境下達到毛髮新生的效果。本研究選用聚二甲基矽氧烷（Polydimethylsiloxane, PDMS）作為材料，由於其本身良好的生物相容性及細胞不易貼附的特性，將真皮乳頭細胞培養在已塗覆PDMS的96孔盤內便能使其自行聚集形成微球組織，再加上表皮細胞在外層包覆並透過免疫螢光染色確保其仍保有促進毛囊新生能力。為了提供適當的生長骨架，以膠原蛋白（collagen type I）與細胞微球組織混合使膠原蛋白包覆著微球成膠，並植入裸鼠體內觀察其發育形態，以證明本實驗設計之真皮乳頭細胞微球能促進毛囊的生成及毛髮再生，並在未來有應用於毛囊重建之組織工程的潛力。

Alopecia is a disturbing problem effecting men and women of all ages. Because the syndrome directly affects the appearance, cause physiological impacts on patient. The treatment for alopecia involves either hair transplant, pharmacotherapy or phototherapy. But these treatment options are fraught with problems of cost, side effects, and treatment effect is unstable. Recently, with the development and trend of tissue engineering, more and more scholars study on hair follicle regeneration. Cell-based therapies have focused on the dermal papilla cell. However, many obstacles exist, inducing ability and maintenance of dermal papilla productivity after several passages of culture. The natural hair development process depends on a series of interactions between dermal papilla cells and epidermal cells. Thus, the DP cells and epidermal cells are cultured together to form a three-dimensional spheroid structure for promote cells interaction and retain the ability to induce hair regeneration.

Therefore, the aim of this study was to culture dermal papilla cells and epidermal cells to form microsphere tissues, which are high-throughput and can be produced in uniform-size manner, and to investigate whether these microsphere tissues could induce hair regeneration. Polydimethylsiloxane (PDMS) was selected as the material. Due to its good biocompatibility and low cell adhesion, dermal papilla cells were cultured in 96-well discs coated with PDMS to form micro spheroids. Moreover, epidermal cells were added to form “core-shell” structure, and immunofluorescence staining was used to ensure that they still retained the ability to promote hair follicle regeneration. On the other hand, in order to provide an appropriate scaffold, we choose collagen type I as biomaterial scaffolds. Microspheres were collected and mixed with collagen solution to form a hydrogel and transplanted into nude mice, then observe their morphology to prove that this experimental design can induce hair regeneration and has potential application in hair follicle reconstruction in the future.

致謝 I
摘要 II
ABSTRACT III
目錄 V
圖目錄 VII
第一章 序論 1
1.1毛髮與毛囊 1
1.2 毛囊生長週期 2
1.3禿髮 3
1.4禿髮治療手段 3
1.4.1 藥物治療 3
1.4.2 光治療 4
1.4.3 自體頭髮移植 4
1.5 毛囊組織工程--毛囊重建 4
1.5.1 毛囊幹細胞 4
1.5.2 三維細胞培養（3D culture） 5
1.5.3 共培養系統（co-culture） 5
1.6 動物實驗模型（IN VIVO） 6
第二章 研究概述 8
2.1 研究背景 8
2.2 研究動機與目的 8
2.3 方法簡述 9
2.4 實驗流程圖 9
第三章 實驗材料與方法 10
3.1 實驗藥品 10
3.2 實驗儀器 12
3.3 低貼附性96-WELL之製備 13
3.4 人類毛囊真皮乳頭細胞及角質形成細胞之培養 13
3.5 新生小鼠角質細胞之分離與培養 14
3.6 角質形成細胞KERATINOCYTE與真皮乳頭細胞（DP CELL）共培養系統 14
3.7 CORE-SHELL細胞培養 15
3.8 PKH26細胞紅螢光染色 16
3.9 PKH67細胞綠螢光染色 16
3.10 細胞生存力染色 16
3.11 免疫細胞化學（IMMUNOCYTOCHEMISTRY, ICC）與鹼性磷酸酶染色 17
3.12 支架製備— COLLAGEN TYPE I SCAFFOLD 19
3.13 動物實驗—皮下細胞注射（SUBCUTANEOUS INJECTION）與PATCH ASSAY 19
3.14 組織切片染色 20
第四章 結果與討論 21
4.1 新生小鼠角質細胞的培養及形態觀察 21
4.2 人類真皮乳頭細胞培養至細胞低貼附性之96孔盤的形態觀察 22
4.3 真皮乳頭細胞與角質細胞共同成球 23
4.4 真皮乳頭細胞微球組織活性分析 25
4.5 IN VITRO培養下真皮乳頭誘導毛囊再生之能力 26
4.6 真皮乳頭微球組織在活體內誘導毛囊生成之能力驗證 30
第五章 結論 32
第六章 參考文獻 33

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