近幾年研究結果指出毛囊幹細胞可能存在毛囊的髮凸(bulge)結構裏，所以也被命名為髮凸幹細胞(bulge stem cells)，經由動物體內的細胞系追蹤(lineage tracing)實驗或皮膚移植實驗結果顯示，毛囊幹細胞具有形成角質細胞以修復受傷表皮、分化成皮脂腺細胞以及再生毛髮的多潛能的分化能力。近期研究也發現毛囊幹細胞可能表現特殊細胞表面標誌如CD34、CD49f等，因此可基於此特性而藉由流式細胞儀從皮膚分離出來，然而並未有研究確切指出毛囊幹細胞可在體外環境(in vitro)中培養增生並可維持其多潛能分化的特性，進一步地並可在植入皮膚組織而後仍可以長出毛髮。因此本論文目標在嘗試建立體外培養環境來提供毛囊幹細胞進行增生及分化，並探討各種調控因子對於毛囊幹細胞在毛囊分化過程中的影響。由目前結果指出，在E13.5與E18.5鼠胚胎纖維母細胞(mouse embryonic fibroblast)的共同培養下可模擬毛囊發育的微環境(microenvironment)，而維持成鼠毛囊幹細胞未分化的特性並可在體外環境增生，相反地與初生鼠皮膚纖維母細胞(dermal fibroblasts; DF)共同培養下，成鼠毛囊幹細胞容易分化為角質細胞，顯示不同滋養層細胞可能藉由分泌調控因子的不同而影響幹細胞生長與分化狀態，進ㄧ步地如將培養於E18.5 胚胎纖維母細胞上之毛囊幹細胞注射入初生鼠之上唇皮膚的真皮層中作體外器官培養(ex vivo organ culture)，在四星期後，發現會有細毛產生，此結果說明在E18.5 胚胎纖維母細胞的共同培養環境，除能促進毛囊幹細胞增生外，還能維持其分化特性，因此未來可進一步利用這樣的培養系統來探討微環境中的各種因子對於毛囊幹細胞分化及自我更新的調控。

Recent studies using in vivo engraftment studies have revealed that hair follicle stem cells, which reside in the bulge of the hair follicle, could differentiate into multiple cell types including, sebaceous glands, entire hair follicle and epidermis. Moreover, several groups have successfully isolated hair follicle stem cells recently with specific cell surface markers such as CD34 and CD49f. However, there is no evidence showing that isolated hair follicle stem cells could maintain its differentiation capability in vitro. Therefore, in the current studies, , with the utilization of two-step trypsin digestion method and fluorescence-activated cell sorting (FACS)based on the expression of CD34 and CD49f, we have established a method to isolate hair follicle stem cells from neonatal/adult mice.. When coculturing isolated hair follicle stem cells with different feeder cells including mouse embryonic fibroblasts (MEFs) isolated from various embryonice stage , neonatal dermal fibroblasts (DFs), and 3T3 cells, we found that inE13.5 and E18.5 MEF-cocuturing condition, stem cell colonies were formed with appearance of undifferentiated cell morphology. In contrast, when hair follicle stem cells grown onto DF together with EGF and calcium, stem cells differentiated into keratinocyte-like cells. Furthermore, injection of stem cell colonies obtained from E18.5 MEF-co-culturing condition into neonatal upper-lip, , we found that fine hairs were grown from lips after four-week’s culture. In summary, our results suggest that embryonic fibroblast cells can possibly provide a suitable microenvironment to expand and to maintain the multipotency of adult hair follicle stem cells in vitro.

目錄
中文摘要.............................................................1
英文摘要.............................................................3
中英名詞縮寫對照表.....................................................4
第一章 緒論..........................................................5
1.1. 前言.........................................................5
1.2. 毛囊發育與型態發生.............................................6
1.3. 毛髮生長週期..................................................9
1.4. 毛囊形態發生與出生後更新週期中之分子調控機轉.....................11
1.4.1. 真皮乳突....................................................12
1.4.2. Wnt signaling..............................................13
1.4.3. Bone morphogenetic proteins (BMP) signaling................14
1.4.4. Hoxc13.....................................................16
1.5. 毛囊幹細胞...................................................18
1.6. 實驗動機....................................................21
1.7. 實驗目的....................................................23
第二章 實驗材料與方法................................................24
第三章 實驗結果.....................................................33
第四章 討論.........................................................42
參考文獻............................................................50
圖..................................................................59
表..................................................................88

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