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研究生： 羅韻綺
Lo, Yun-Chi
論文名稱： 以體外皮膚培養系統研究微環境對於毛囊之初始發育
Effects of microenvironments on hair follicle initiation using an organotypic skin culture system
指導教授： 黃玲惠
Huang, Lynn L.H.
學位類別： 碩士
Master
系所名稱： 生物科學與科技學院 - 生物科技研究所
Institute of Biotechnology
論文出版年： 2010
畢業學年度： 98
語文別： 中文
論文頁數： 109
中文關鍵詞： 頭髮缺失 、毛囊體外培養再生系統 、第一型膠原蛋白 、真皮層等價物
外文關鍵詞： alopecia, in vitro organotypic skin culture systems, type I collagen, dermal equivalent
相關次數： 點閱：175  下載：8 
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    • 頭髮缺失並不是威脅生命的重要疾病，但對人類的社交及心理所造成的影響則是無庸置疑的。毛囊再生需要經過表皮層細胞與真皮層細胞之間的交互作用，並牽涉到多個分子訊息的傳遞。將皮膚中取得的表皮層細胞及真皮層細胞共同培養在小鼠及人類的皮膚裡，經過交互作用後，可以誘導生成新的毛囊。目前在研究治療毛囊缺失或毛髮生長的藥物面臨一個很大的困境，也就是缺乏一個有效且完善的3D體外篩選平台，所以發展毛囊體外培養再生的系統以篩選治療毛髮缺失藥物的需求實是刻不容緩。我們想要利用3D體外毛囊培養系統模擬皮膚的環境來促進體外毛囊發育及生長；3D體外毛囊培養系統的一部分是將從豬皮分離所得的第一型膠原蛋白與新生小鼠的真皮層細胞混合作為真皮層等價物，然後將新生小鼠的表皮層角質細胞，經過這些細胞的聚合、增生及分化，得以再生出新的毛囊。此外，我們會在適當時間加入hydrocortisone、insulin、progesterone以及WNT10B蛋白質，藉由這些對毛囊發育及生長有影響力的分子，可以提高毛囊再生的效率。我們的目標是建立老鼠體外毛囊再生的模型，並藉由這個模型進一步了解毛囊的生物特性。

    Although alopecia is not life threatening, their profound impact on social interactions and on patients’ psychological well being is undeniable. Hair follicle regeneration is a complicated process of epithelial-mesenchymal interaction involving various molecular signals. By mixing cellular components from epidermal and dermal parts of the skin, researchers had established in vivo hair growth models in mice and in human. However, the search for agents that induce hair growth has been severely handicapped by the lack of satisfactory three-dimensional (3D) in vitro screening systems that sufficiently mimic important epithelial-mesenchymal interactions as they occur in human hair follicles. Therefore, pragmatic in vitro organotypic skin culture systems are badly needed. We use a 3D collagen culture system; one part is a dermal equivalent consisting of type I collagen from pig skin and dermal cells from neonatal mice, another part is a layer of epidermal keratinocytes from neonatal mice put on the dermal equivalent, to provide an environment for hair development and growth. By modifying the volume and the collagen concentration of the dermal equivalent and increasing the cell density, we try to optimize the efficiency for cell-cell interaction. We introduce molecules which play an important role in hair follicle development and maturation, such as hydrocortisone, insulin, progesterone into the culture medium to promote the hair regeneration ability. In our system, we observe cells within the culture system go through interaction, proliferation, differentiation and assembly to form spheroid cell aggregates. In another way, using WNT10B proteins with hair inducing ability, we want to increase the number of regenerated hair follicles. Our ultimate goal is to develop an in vitro system to regenerate completely organized and functional hair follicles. The system will also help us to understand the biology of hair follicles.

    中文摘要.........................2 英文摘要.......................3 誌謝.................................5 目錄.................................6 表目錄.............................12 圖目錄.............................13 符號及縮寫..............................15 第一章 研究背景與目的 1.1毛囊之簡介...............................16 1.1.1毛囊之解剖結構........................16 1.1.2毛囊之類型.......................17 1.1.3毛囊之細胞類型........................17 1.1.4毛囊之發育與形態發生.....................18 1.1.5毛囊之生長周期........................21 1.2毛囊發育及毛囊生長周期相關細胞及生長因子之簡介....22 1.2.1與毛囊發育及調控毛囊生長周期相關之細胞............22 1.2.2分佈於毛囊附近各種細胞之表面標誌...............23 1.2.3發育中毛囊之分子標誌.....................24 1.2.4影響毛囊發育及調控毛囊生長周期之因子.......24 1.3頭髮缺失疾病簡介............................27 1.3.1雄性遺傳脫髮之症狀................28 1.3.2雄性遺傳脫髮之成因................28 1.3.3雄性遺傳脫髮之療法................29 1.4毛囊在再生醫學上的應用性.....................31 1.4.1毛囊培養再生之方法................32 1.4.2毛囊的體內培養再生之文獻回顧..............33 1.4.3毛囊的體外培養再生之文獻回顧..............35 1.5實驗目標..........................40 1.6實驗設計..........................40 1.6.1新生小鼠皮膚毛囊、胞外基質及細胞分佈.......40 1.6.2小鼠毛囊相關細胞之分離.................40 1.6.3細胞類型之分析及鑑別.....................41 1.6.4細胞之標定.......................41 1.6.5小鼠體外毛囊培養再生模型之建立..........41 1.6.6小鼠體外毛囊培養再生模型細胞之分析及鑑定........42 1.6.7WNT10B蛋白質之表現與純化..................42 第二章 藥品、儀器與方法 2.1藥品..................................44 2.1.1細胞培養...........................44 2.1.2細胞染色...........................44 2.1.3免疫組織化學染色....................44 2.1.4PCR試劑...........................44 2.1.5純化質體DNA試劑................45 2.1.6實驗所用之抗體........................45 2.2儀器..................................45 2.3方法..................................45 2.3.1新生小鼠皮膚毛囊、胞外基質及細胞分佈.......45 2.3.1.1組織包埋及石蠟切片.................45 2.3.1.2蘇木素-伊紅染色(H&E staining) ...............46 2.3.1.3艾爾遜藍染色(Alcian Blue staining)...........46 2.3.1.4免疫組織化學染色(Immunohistochemical staining).............................46 2.3.2小鼠毛囊相關細胞之分離.................47 2.3.2.1小鼠表皮角質細胞之分離..................47 2.3.2.2小鼠真皮纖維母細胞之分離..............47 2.3.2.3小鼠毛囊角質細胞之分離..................47 2.3.2.4小鼠脂肪前細胞之分離.............48 2.3.3細胞染色 .......................48 2.3.4細胞標定(PKH26 labeling) .............48 2.3.5小鼠體外毛囊再生培養系統.............48 2.3.5.1膠原蛋白體積....................49 2.3.5.2膠原蛋白濃度....................49 2.3.5.3細胞密度............................49 2.3.5.4小鼠體外毛囊再生培養系統之建立...........49 2.3.6小鼠體外毛囊再生培養系統細胞之分析及鑑定........51 2.3.7人類WNT10B蛋白質之表現與純化 ...............52 2.3.7.1人類WNT10B蛋白質序列引子之設計 ............52 2.3.7.2以PCR製造帶有合適限制酶切點及3’tag的產物.............................52 2.3.7.3純化PCR反應之產物 .............53 2.3.7.4利用限制酶將人類wnt10b序列接合至表現載體.............................53 2.3.8選殖穩定表現人類WNT10B蛋白質的細胞株 ........53 2.3.9WNT10B蛋白質之功能分析.............54 2.3.9.1短暫轉染及報導基因表現分析..........55 2.3.9.2 穩定轉染及報導基因表現分析..........57 2.3.9.3抑制脂肪前細胞的分化.............58 2.3.9.4西方墨點法........................59 第三章 實驗結果 3.1實驗動物..........................60 3.2新生小鼠皮膚之毛囊、胞外基質及細胞分佈...........60 3.2.1蘇木素-伊紅染色(H&E staining) ..............60 3.2.2艾爾遜藍染色(Alcian Blue staining)..........60 3.2.3免疫組織化學染色(Immunohistochemical staining)....61 3.2.3.1真皮纖維母細胞................61 3.2.3.2真皮幹細胞........................61 3.2.3.3真皮乳突細胞....................61 3.3小鼠毛囊相關細胞之分離................62 3.3.1小鼠表皮角質細胞(Mouse epidermal keratinocytes).................................62 3.3.2小鼠真皮纖維母細胞(Mouse dermal fibroblasts) ....62 3.3.3小鼠毛囊角質細胞(Mouse hair follicle keratinocytes) .................................62 3.3.4小鼠脂肪前細胞(Mouse preadipocytes) ...........62 3.4免疫細胞染色...........................62 3.4.1真皮幹細胞.......................63 3.4.2真皮乳突細胞............................63 3.5細胞標定（PKH26 labeling） .................63 3.6小鼠體外毛囊再生培養系統.....................63 3.6.1膠原蛋白體積............................63 3.6.2膠原蛋白濃度............................64 3.6.2.1膠原蛋白濃度3mg/ml，未經氣-液介面培養，培養三天...........................64 3.6.2.2膠原蛋白濃度6mg/ml，未經氣-液介面培養，培養三天...........................64 3.6.2.3膠原蛋白濃度3mg/ml，未經氣-液介面培養，培養七天...........................64 3.6.2.4膠原蛋白濃度6mg/ml，未經氣-液介面培養，培養七天...........................64 3.6.3細胞密度...........................65 3.7 WNT10B蛋白質之表現與純化.................65 3.7.1人類WNT10B蛋白質之表現與純化 ...............65 3.7.1.1人類WNT10B蛋白質序列引子之設計 ............65 3.7.1.2以PCR製造帶有合適限制酶切點及3’tag的產物.............................66 3.7.1.3純化PCR反應之產物 .............66 3.7.1.4利用限制酶將人類wnt10b序列接合至表現載體.............................66 3.7.2選殖穩定表現人類WNT10B蛋白質的細胞株........66 3.7.2.1測試G418對實驗中使用的293細胞最佳篩選濃度.............................67 3.7.2.2將人類wnt10b序列轉殖至293細胞 .......67 3.7.2.3選殖穩定表現人類WNT10B蛋白質的細胞株.............................67 3.8 WNT10B蛋白質之功能分析.....................68 3.8.1短暫轉染及報導基因表現分析..................68 3.8.2穩定轉染及報導基因表現分析..................69 3.8.2.1TCF Reporter Plasmids序列引子之設計 ............69 3.8.2.2以PCR製造帶有合適限制酶切點及3’tag的產物.............................70 3.8.2.3 純化PCR反應之產物 .............70 3.8.2.4利用限制酶確認TOPflash序列接合至TOPO載體.............................70 3.8.3 對於脂肪前細胞脂肪分化之影響..............70 3.8.3.1脂肪前細胞之脂肪分化.............70 第四章 討論 4.1老鼠體外毛囊再生培養系統之建立..................93 4.2未來研究方向...........................94 第五章 參考文獻...........................108

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