- 學位論文
人類毛囊的發育與Wnt分子對皮膚形成的調控
Human Hair Follicle Development and Wnt Molecules Regulate the Skin Morphogenesis
摘要
器官的形成，猶如一支交響樂，有許多樂章共同譜成，包括：細胞命運的決定、誘導、極化、模組形成、附著、增生、分化、凋亡等。本研究首先探討人類皮膚附屬器毛髮，胎生期發育的過程中，增生、凋亡與分化扮演的角色。毛髮形成初期，增生與凋亡相伴進行，凋亡發生於生長區，如毛根外鞘及結締組織層，以雕刻或調控生長區；毛髮形成後期，分化與凋亡相伴進行，凋亡發生於特定部位，以製造空間，分離緊臨的組織，如companion layer及毛根內鞘；毛髮形成後要穿出皮表，凋亡是形成hair canal最重要的機轉，凋亡產生的同時，canal周圍細胞最終分化以形成角質層，當毛髮暴露於皮表時，毛囊間皮膚也同時完成了保護功能的角質層。凋亡現象在各時空精確地出現，與增生、分化間達成平衡，完成了皮膚附屬器特有的型態，並成為獨立的功能單位。在器官發育的過程中，有幾個訊息分子家族，反覆地參與其中，如Wnt, BMP, FGF, Shh等，本研究以雞胚胎為模式，探討Wnt在皮膚形成各階段所扮演的角色。皮膚的形成是具有層次性與連續性的過程。首先，來自中胚層的真皮母細胞移行到未來形成表皮的外胚層下方，形成體表大塊的皮膚區域（稱為巨大模組），之後在每一個大區域上再形成個別具有特定形狀與大小之皮膚附屬器（稱為微小模組）。DKK1﹙Wnt的拮抗分子﹚的過度表現可以抑制羽毛區域真皮的形成，與區域中羽毛胚的形成，顯示了Wnt在皮膚形成的重要性。本論文以羽毛為模式，探討不同的分子在皮膚形成的的不同階段具有的功能。Wnt1 、3a、 5a和 11在羽毛區塊形成的初期均勻的表現在表皮﹙Wnt1 、3a、 5a﹚和真皮﹙Wnt11﹚。之後，在羽毛胚形成時，則以兩種不同的模式增強表現：Wnt1和Wnt3a侷限於羽毛胚的表皮，及之後增長羽毛胚遠端的表皮；Wnt5a和Wnt11則分別侷限於區塊之間及羽毛胚之間的表皮與真皮，並且之後出現在增長羽毛胚遠端的真皮。我們以RCAS病毒攜帶特定Wnt基因打入E2/E3雞胚胎，影響Wnt分子表達的方式，來探討Wnt對皮膚區塊形成的影響。以E7真皮細胞感染RCAS病毒的重組皮膚體外培養，探討Wnt對羽毛胚形成的影響。Wnt1抑制真皮的形成及減小羽毛脊椎區域的大小，但對個別羽毛胚形成具有促進作用。Wnt3a促進真皮形成，擴大羽毛脊椎區域的範圍，減少羽毛胚之間的距離，產生遠端不會變細的巨大羽毛胚。Wnt11和dominant negative Wnt1增加羽毛胚之間的距離並且產生稀少且細小的羽毛。在真皮纖維母細胞的培養實驗中，Wnt1和Wnt3a刺激細胞的增生，並激活beta-catenin訊息傳導路徑。Wnt11抑制細胞的增生，但促進細胞的移行。Wnt5a和Wnt11可激活JNK路徑。因此，本研究顯示Wnt分子在真皮、皮膚區域，附屬器間的距離，及附屬器生長形狀等，各階段形成的調控上，扮演了正向或負向調節的功能。本研究的發現，將有助於人類皮膚疾病如形成異常與癌化等，機轉的了解，與未來治療與再生醫學的應用。
關鍵字
none並列摘要
Organogenesis is orchestrated by many important events, including fate specification, induction, polarization, pattern formation, adhesion, proliferation, differentiation, apoptosis, etc. We studied the role of proliferation, apoptosis and differentiation in developing human hair follicles. Apoptosis within the proliferative zone, such as outer root sheath and connective tissue sheath, may help to shape the localized growth zone. Apoptosis in companion layer and inner root sheath is required to release the hair shaft from the follicle wall. Apoptosis results in the epidermal and subepidermal hair canal formation to release hair unto the skin surface, which happens in parallel with the terminal differentiation of interfollicular epithelium. There are several signaling molecules repeatedly involved in the organogenesis, including Wnt, BMP, FGF, Shh, etc. Here we studied the role of Wnt in the hierarchical morphogenesis of skin by chicken embryo model. Skin morphogenesis occurs in succession, with one level unfolding after another. First dermal precursors migrate to the subectoderm. The presumptive dermis forms distinct skin regions over the body (macropatterning). Individual skin appendages form within each region (micropatterning) with a particular shape and size. Ectopic DKK1 expression inhibited dermis formation in feather tracts and individual feather buds, implying the importance of Wnts, and prompted the assessment of individual Wnt functions at different morphogenetic levels using the feather model. Wnt 1, 3a, 5a and 11 initially were expressed moderately throughout the feather tract, then were up-regulated in restricted regions following two modes: Wnt 1 and Wnt 3a became restricted to the placodal epithelium, then to the elongated distal bud epidermis. Wnt 5a and 11 intensified in the inter-tract region and inter-primordia epidermis and dermis respectively, then appeared in the elongated distal bud dermis. Their role in feather tract formation was determined using RCAS mediated misexpression in ovo at E2/E3. Their function in periodic feather patterning was examined by misexpression in vitro using reconstituted E7 skin explant cultures. Wnt 1 reduced spinal tract size, but enhanced feather primordial size. Wnt 3a increased dermal thickness, expanded the spinal tract size, reduced the interbud domain spacing, and produced non-tapering "giant buds". Wnt 11 and dominant negative Wnt 1 enhanced interbud spacing, and generated fewer and thinner buds. In cultured dermal fibroblasts, Wnt 1 and Wnt 3a stimulated cell proliferation and activated the canonical beta-catenin pathway. Wnt 11 inhibited proliferation but stimulated migration. Wnt 5a and 11 triggered the JNK pathway. Thus distinctive Wnts have positive and negative roles in regulating the formation of the dermis, tracts, interbud spacing and growth and shaping of individual buds. The knowledge of developmental biology may help the assessment of human skin diseases, including malformation and carcinogenesis, and also imply the application in treatment and regeneration.