臺灣博碩士論文加值系統

皮膚附屬器，特別是毛囊與汗腺，在維持哺乳類個體環境的恆定非常重要。其中毛囊發育及週期生長的調控，與身體諸多組織器官(如角膜、唾腺等)一樣，皆透過上皮(epithelial)與間質(mesenchymal)細胞精密的交互作用來達成。毛囊生成，不管是在胚胎發育時期，還是在出生後，都是經由這種上皮-間質兩個胚層作用發展而出的微器官。這個微器官，會隨著年齡老化而逐漸減少導致禿髮。臨床上最常見的禿髮，是因先天基因與後天環境的影響所導致的雄性禿髮，其毛囊器官逐漸變小，生長期縮短，咸認是一種器官老化的過程，特別是在某些年輕的個體，此老化過程明顯提前；另一類常見禿髮，肇因於發炎性的疾病(如紅斑性狼瘡)所引起毛囊結構纖維化而導致禿髮。不管是哪一種禿髮，對患者的情緒與社交功能都有很大的影響。然而，目前針對禿髮的治療，不管是利用口服或外擦藥物，或是經由手術移植毛囊單位(植髮)，都沒有再生「新」的毛囊，也就是說，當前的治療仍舊侷限於挽救或重分佈既有毛囊的框架中，此類治療對於重度禿髮的患者，無法帶來令人滿意的成果。

在毛囊生成的過程中，間質真皮乳突細胞(dermal papilla cells; DP cells)調控毛髮的生長與週期變化，是透過對毛囊上皮角質細胞(keratinocytes)的作用而來。真皮乳突細胞在毛囊再生扮演居中重要的誘導功能，將上皮組織的角質細胞，誘導分化為毛囊型的上皮角質細胞，進一步發展出毛囊結構，並生出終產物：髮幹(hair shaft; HS)。為了增加新生毛囊的數目，目前的發展已經推進到研究者可以在體外透過大量培養成鼠的真皮乳突細胞來誘導上皮角質細胞再生新髮。在動物實驗模型上，一則使用具有高毛囊誘導(folliculogenic)能力的新生鼠表皮細胞，與大量培養的成體真皮乳突細胞作用產生新髮；另一則使用成體誘導幹細胞(induced pluripotent stem cells)分化而成的上皮細胞，與新生鼠的真皮細胞作用生髮。不管是哪一種可用的毛髮再生動物模型，都取巧一端使用新生個體較具有發育潛能的間質或上皮細胞。當前最常使用的組合，是透過培養的成體真皮乳突細胞，混和新生鼠之表皮細胞來再生新髮。這種使用新生鼠的表皮細胞作為毛囊誘導的上皮端，在應用層面並不可行。因為透過毛囊新生治療禿髮，終究是要取得禿髮個體的真皮乳突細胞，與來自同一個體的表皮角質細胞，透過上皮-間質的交互作用，從而再生新髮。

既然來自真皮的乳突細胞已知可經過大量體外培養仍維持其毛囊誘導能力，該解決的部分變成是如何讓成體的表皮細胞也可以大量培養並維持其毛囊生成能力。可惜的是，經過體外培養繼代的成體表皮細胞，很快失去了被誘導長毛的能力。本研究的設計，便是試圖建立一個全部利用成鼠細胞(包括真皮乳突細胞與表皮角質細胞)經過培養增量，來再生新髮的模型，並討論如何讓成鼠的表皮角質細胞變得像新生鼠般，具有高度誘導毛囊化的能力。雖然成體上皮角質細胞經過培養無法被成功誘導再生新髮，但本研究透過直接將體外培養的成體角質細胞與具有毛囊誘導能力的真皮乳突細胞，共置於培養環境中，讓細胞可以直接接觸，重現胚胎發育的過程中，間質-上皮兩個胚層的交互作用。此方法成功地重現成體角質細胞的毛囊化基因標記，且在動物模型確實誘導再生新髮。

本研究的目的，在於使用成鼠細胞來實現毛囊新生，並對於可再度往毛囊分化的成體角質細胞進行功能性分析及其調控機轉探討。希望透過更了解毛囊這個皮膚附屬器的再生科學，建立穩定模型以供未來發展再生新髮的治療模式，帶來更多臨床上應用價值。

Like other organs including cornea and salivary gland, the development and cyclic growth of hair follicle (HF) is also governed by epithelial-mesenchymal interaction. Dermal papilla (DP) cells, the HF mesenchymal cells, have been shown to regulate hair growth and cycles, at least in part through its interaction with follicular epithelial cells or stem cells. Whenever the interaction is interrupted or disturbed, affected individual will develop various hair diseases. The most common hair loss, androgenetic alopecia (AGA), results from follicular miniaturization and shortened growth period in hair cycles. The hair organ miniaturization in AGA is considered to be a normal aging process. However, for those who are young with early development of AGA, the process seems to be “premature” that might have something to do with the follicular environment and hormonal change. What leads to the premature aging is not yet elucidated.

Current therapeutic options for hair loss are either surgical redistribution of scalp hairs, or medical treatments including oral or topical preparations. These clinically available surgical or non-surgical treatments do not increase patient’s follicular units. To ultimately solve the problem of hair loss, the best way is to generate new hair follicles.

Resembling embryonic hair morphogenesis, adult hair neogenesis involves delicate signal reciprocations between follicular epidermal cells and mesenchymal DP cells. New and considerable HFs can be generated by the advances in the techniques to combine inductive cultured DP cells and competent epidermal stem cells. Most regeneration models prefer to use cultured adult DP cells and “newborn” epidermis, taking the advantage of the multipotency of the newborn epidermal cells. This strategy is not applicable in the clinical aspect since the end goal of hair regeneration is to take cells from alopecia individuals who are mostly adult. Current hair regeneration models remain unable to efficiently expand folliculogenic adult epidermal cells. Only with large number of competent adult epidermal cells can we eventually apply the hair regeneration technique to adult individuals.

To study the effect of in vitro epidermal-mesenchymal interaction and its impact on hair follicle regeneration, we cultured keratinocytes and DP cells into the same dish before they are used in further hair regeneration experiments. High passage rat epidermal cells successfully responded and formed hair shafts in the initial experiments. The in vitro epidermal-mesenchymal interaction distinctly changes epidermal cells to express elevated hair-specific genes. Our data showed the cultivated adult cells recapitulate the epidermal-mesenchymal interaction that induces the high passage epidermal cells to adopt the follicular fate and head to hair follicle differentiation. This acquired hair induction ability provides a new field for bioengineered hairs that not only the dermal cells but also the epidermal cells can be taken for population expansion before hair regeneration. This finding simply announces the possibility of removing one single adult hair, separately expanding dermal and epidermal cell numbers, and then coculturing these considerable amounts of cells before finally mixing them to form numerous new hairs. Successful development of this model and clarification of the underlying mechanism may lead to future large-scale hair production by bioengineering these adult follicular cells.

口試委員會審定書.........I
中文摘要.........II
ABSTRACT.........IV
LIST OF ABBREVIATIONS.........VII
CONTENTS.........VIII
LIST OF FIGURES.........XIII
LIST OF TABLE.........XV
Chapter 1 Introduction.........1
1.1 Development of a Hair Follicle.........2
1.1.1 Structure of the Hair Follicle.........4
1.1.1.1 Hair Follicle Epidermal Layers.........4
1.1.1.2 Hair Follicle Bulb.........4
1.1.1.3 Hair Follicle Stem Cell Niche: the Bulge.........5
1.1.1.4 Sebaceous Gland.........5
1.1.1.5 Hair Follicle Dermal Cells.........5
1.1.2 Hair Cycling.........9
1.1.2.1 Anagen.........9
1.1.2.2 Catagen.........9
1.1.2.3 Telogen.........10
1.2 Hair Follicle Formation Mechanisms: Morphogenetic Switch or Neogenesis.........12
1.3 Roles of Epidermal Component in Hair Follicle Induction.........15
1.4 Which Type of Keratinocytes Is the Target in Response to Dermal Signals?.........18
1.4.1 Location of Epidermal Stem Cells.........18
1.4.2 Plasticity of Follicular Epithelial Stem Cells and Interfollicular Epidermis.........19
Chapter 2 Theoretical Basis and Hypothesis.........23
2.1 Tissue Engineering for Hair Follicle Regeneration.........23
2.1.1 Hair Regeneration Model: Patch Model.........24
2.1.2 Hair Regeneration Model: Chamber Model.........24
2.1.3 Hair Regeneration Model: Sandwich Model.........28
2.1.4 Hair Regeneration Model: Wound Model.........28
2.2 Production of Trichogenic Dermal and Epidermal cells.........29
2.2.1 Generation of Trichogenic Dermal cells.........29
2.2.2 Generation of Trichogenic Epidermal cells.........30
2.3 Production of Trichogenic Epidermal cells with Cultured Adult Cells via In Vitro Epithelial-Mesenchymal Interaction: the Hypothesis.........32
2.3.1 Revival of Cellular Ability.........32
2.3.2 In Vitro Epidermal-Mesenchymal Interactions Facilitate Cultured Epidermal Cells to Revive Trichogenicity?.........32
2.4 Production of Trichogenic Epidermal cells with Cultured Adult Cells via Upregulation of Wnt Signaling Pathway: the Hypothesis.........35
2.4.1 The Role of Wnt/β-Catenin in Transdifferentiating Follicular Keratinocytes.........35
2.4.2 Can Upregulation of Wnt Signaling Pathway Facilitate Cultured Epidermal Cells to Revive Trichogenicity?.........36
2.5 Summary of Hypothesis and Goal of the Study.........39
Chapter 3 Materials and Methods.........40
3.1 Animals.........40
3.2 Cell culture and treatments.........40
3.3 Media.........42
3.4 Preparation of cocultured cells.........42
3.5 Patch assay for hair follicle regeneration.........43
3.6 Histology examination.........43
3.7 Immunofluorescence staining and microscopy.........44
3.8 Preparation of cDNA and quantitative PCR.........45
3.9 SDS-PAGE and Western blot analysis.........47
3.10 Statistical analysis.........48
Chapter 4 Results.........49
4.1 High passage adult keratinocytes retained the same morphology and reproductive capacity as primary cells, but not the ability of hair formation.........49
4.2 Coculturing with DP cells enabled high passage adult keratinocytes to regain hair forming ability.........50
4.3 Follicular gene expression was upregulated in cocultured epidermal cells.........52
4.4 Wnt/β-catenin signaling was activated in the cocultured epidermal cells.........53
4.5 Coculture resulted in β-catenin accumulation in adult high passage epidermal cells, and Wnt/β-catenin signaling was upregulated in both mRNA and protein levels.........54
Chapter 5 Discussion.........57
Chapter 6 Conclusions.........62
REFERENCE.........76

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