臺灣博碩士論文加值系統

皮膚為人體最大的器官，具有高複雜性與高功能性，也具有許多附屬器官，而毛囊為其中一個重要的附屬器官。毛囊是一個高自主性的微小器官，具有毛囊幹細胞與自我生長週期，型態會隨著生長週期的不同而有所變化。因此，毛囊本身的生理調控，以及在受到外界環境刺激後的生理調控與修復機制，以及與環境之間的溝通與響應，都成為再生醫學研究的範疇，使得毛囊成為再生醫學研究重要的實驗研究模型。
休止期時期的毛囊會受到許多引發因子(initiator)的影響而活化，細胞增生而逐漸生長進入生長期狀態，而這些引發因子的相關研究蓬勃發展。本研究目標希冀能探索毛囊感應到外界刺激後活化，而從休止期狀態生長進入生長期。本研究以十二烷基硫酸鈉(Sodium dodecyl sulfate, SDS)作為皮膚刺激物，並在休止期時期時給予刺激，誘發刺激性接觸性皮膚炎。在SDS連續刺激下，休止期狀態的毛囊，其毛囊幹細胞活化而進行細胞增生，毛囊逐漸生長而過早地進入生長期。在SDS連續刺激後，引發許多免疫細胞浸潤到毛囊周圍區域，包括嗜中性白血球和巨噬細胞。之後，我們將巨噬細胞耗竭，而不是嗜中性白血球耗竭，可以明顯地阻擋SDS刺激後休止期毛囊過早進入生長期，此結果可以得知巨噬細胞在SDS刺激而誘發休止期毛囊活化生長而提早進入生長期中扮演重要角色。
本研究也指出血管內皮生長因子(VEGF)在SDS刺激後，表現量會明顯地增加，且與巨噬細胞有關聯性。抑制VEGF信號傳導途徑可以延遲SDS刺激所誘發休止期毛囊活化生長而提早進入生長期。
總言之，本研究建立一個休止期狀態的毛囊在響應SDS刺激表皮後，活化生長而過早進入生長期的實驗模型，也發現巨噬細胞和VEGF信號傳導途徑可能在其中扮演重要角色。

Skin, the largest organ in a human body, exhibits high structural complexity for its unique functions. Hair follicles, one of the skin appendages, are a miniorgan with distinct lifelong cyclic growth that are fueled by their resident stem cells. The activity of hair follicle stem cells are tightly regulated by their niche microenvironment for physiological growth as well as regeneration after injuries. Therefore, hair follicles provide an excellent model for the research of stem cell biology.
The telogen hair follicles, activated by many initiators, undergo cell proliferation and become anagen hair follicles, and the researches of these initiators have become popular and well development recently. This study is aimed at exploring how hair follicles react to irritation to interfollicular epidermis to regenerate from telogen to anagen. We employed the irritant sodium dodecyl sulfate (SDS) to induce irritant contact dermatitis during telogen phase. We found that, after SDS treatment, hair follicle stem cells were activated and hair follicles entered anagen prematurely. This was associated with infiltration of immune cells, including neutrophils and macrophages, in the perifollicular environment. Depletion of macrophages, but not neutrophils, prohibited SDS-induced anagen entry, indicating that activated macrophages are essential for hair follicle stem cell activation by SDS treatment.
Molecularly, increased expression of vascular endothelial growth factor (VEGF) was associated with macrophage infiltration. Inhibition of VEGF signaling delayed SDS-induced anagen entry.
In summary, this study shows that hair follicles are able to initiate a protective regenerative reaction in the presence of irritation to interfollicular epidermis. Activation of macrophages and VEGF signaling are involved in activating hair follicle stem cells.

口試委員會審訂書 I
誌謝 II
摘要 III
Abstract IV
圖目錄 VIII
表目錄 XI
第一章、緒論 1
1.1皮膚結構概要 1
1.2毛囊結構與毛囊生長週期概要 2
1.3毛囊生長的調控 5
1.3.1 簡要 5
1.3.2 脂肪細胞(adipocyte) 6
1.3.3 免疫細胞(immune cells) 6
1.3.4 荷爾蒙(hormone) 7
1.4探討誘導毛囊生長 8
1.4.1 拔毛(Plucking) 8
1.4.2 佛波醇-12-十四烷醯-13-乙酸酯 9
1.4.3 傷口(wounds) 10
1.4.4 免疫細胞(immune cells) 11
1.4.5 皮膚刺激物(skin irritants) 12
1.5刺激性接觸性皮膚炎 12
1.6研究動機 14
1.7研究目標 15
第二章、實驗材料與方法 16
2.1建立刺激性接觸性皮膚炎動物實驗模型 16
2.1.1 實驗動物 16
2.1.2 皮膚刺激物一：十二烷基硫酸鈉(sodium dodecyl sulfate, SDS) 16
2.1.3 皮膚刺激物二：巴豆油(croton oil) 17
2.2小鼠背部皮膚組織收集與處理 17
2.3蘇木精─伊紅染色(hematoxylin & eosin staining, H&E staining) 18
2.4免疫螢光染色(immunofluorescence staining) 19
2.4.1 單一抗體免疫螢光染色 19
2.4.2 雙重抗體免疫螢光染色 22
2.5 RNA萃取(RNA isolation) 23
2.6 cDNA合成(complementary DNA synthesis) 24
2.7聚合酶連鎖反應（Polymerase chain reaction, PCR） 25
2.8即時定量聚合酶連鎖反應(real-time quantitative polymerase chain reaction, qPCR) 26
2.9免疫細胞耗竭與細胞激素抑制(Immune cells depletion and factors inhibition) 30
2.10免疫相關轉殖基因小鼠實驗(Immune-related transgenic mice) 32
2.10.1轉殖基因小鼠資訊 32
2.10.2小鼠基因型鑑定(Genotyping) 34
2.11細胞分選(Cell Sorting) 35
2.11.1樣本處理 35
2.12 統計與分析方法(Statistical analysis) 36
2.12.1毛髮生長面積 36
2.12.2表皮厚度與毛囊長度 36
2.12.3巨噬細胞數量統計 36
第三章、實驗結果 38
3.1建立皮膚刺激誘發毛囊再生的B6小鼠實驗模型 38
3.2觀察皮膚刺激誘發毛囊再生的皮膚組織結構變化 45
3.2.1 表皮形態變化 45
3.2.2 毛囊形態變化 46
3.3探討皮膚刺激對於毛囊之影響 49
3.3.1 毛囊幹細胞細胞增生 49
3.3.2 次級毛胚細胞細胞增生 50
3.3.3 誘發休止期毛囊提早進入生長期毛囊 50
3.4探討皮膚刺激誘發的發炎反應與免疫浸潤現象 56
3.4.1 皮膚發炎與免疫浸潤現象 56
3.4.2 白細胞介素1族( Interleukin-1 family, IL-1 family) 56
3.4.3 腫瘤壞死因子-α(Tumor necrosis factor-alpha, TNF-α) 57
3.4.4 嗜中性白血球(Neutrophil) 58
3.4.5 巨噬細胞(Macrophages) 58
3.4.6 血管內皮生長因子(Vascular endothelial growth factor, VEGF) 59
3.4.7 信號轉導及轉錄激活蛋白3(Signal transducer and activator of transcription 3, Stat3) 60
3.5使用抑制劑(inhibitors)或是基因剔除小鼠(knockout mice)探討皮膚刺激誘發的免疫細胞與發炎相關因子在皮膚刺激誘導毛囊再生中的影響 70
3.5.1 IL-1R1基因剔除小鼠(IL-1R1 knockout mice) 70
3.5.2 嗜中性白血球耗竭(Neutrophils depletion) 71
3.5.3 巨噬細胞耗竭(Macrophages depletion) 72
3.5.4 血管生長因子受體抑制劑 74
3.5.5 誘導性角質細胞特定Stat3基因剔除小鼠(Inducible keratinocyte-specific Stat3-disrupted mice ) 75
3.6探討巨噬細胞在皮膚刺激物誘導毛囊再生中的機轉 84
3.6.1 巨噬細胞浸潤 84
3.6.2 巨噬細胞類型 85
3.6.3 巨噬細胞與血管新生 86
第四章、討論 94
第五章、結論 100
第六章、參考文獻 102

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