本文選題：表皮　切入點：干細胞　出處：《南華大學》2014年碩士論文　論文類型：學位論文

【摘要】：研究背景： 表皮干細胞（EpSCs，Epidermal stem cells）自發(fā)現(xiàn)以來一直是研究的熱點，作為皮膚組織的特異性干細胞，具有高度的自我更新能力及多向分化潛能，其不僅維持皮膚日常的新陳代謝及皮膚內環(huán)境穩(wěn)態(tài)，而且與創(chuàng)面修復緊密相連。當受到外界損傷刺激時，EpSCs可主動參與損傷皮膚的修復，通過脫粘附、遷移、增殖，并向表皮終末細胞分化，誘導表皮角質形成細胞向創(chuàng)面爬行，進而覆蓋創(chuàng)面，促進再上皮化，最終達到修復創(chuàng)面的目的。 α-促黑素（α-MSH，α-melanocyte stimulating hormone）最初被人們發(fā)現(xiàn)是因其在促進黑素生成、調節(jié)皮膚色素沉著方面的作用。近年來大量研究證實，α-MSH在抗炎、退熱、抗菌、營養(yǎng)神經、能量平衡、調節(jié)心血管功能、影響動物生物學行為及維持免疫功能相對穩(wěn)定等方面都發(fā)揮了重要的調控作用。α-MSH通過與黑皮素受體（melanocortin receptor，MCR）結合發(fā)揮其生理功能。MCR是一個G蛋白偶聯(lián)型受體，至今已有5種MCR被克隆和定性，皮膚中發(fā)現(xiàn)的主要是MC1R（Melanocortin1receptor，MC1R）、MC2R（Melanocortin2receptor，MC2R）及MC5R（Melanocortin5receptor，MC5R）。已發(fā)現(xiàn)MC1R在黑素細胞、角質形成細胞、皮脂腺細胞、成纖維細胞、朗格漢斯細胞、真皮免疫細胞和內皮細胞等幾乎所有類型的皮膚組織細胞中均有表達，且與這些細胞的關系最為密切。 本實驗從大鼠皮膚基底層分離培養(yǎng)出EpSCs，并進行鑒定；同時檢測EpSCs中α-MSH及其受體MC1R的表達情況，為研究α-MSH對EpSCs的調控作用等后續(xù)實驗打下基礎，以便進一步豐富完善創(chuàng)面修復的基礎理論和臨床應用。 方法： 1．實驗以新生SD大鼠背部皮膚為材料，采用“胰酶兩步消化+IV型膠原差速貼壁法”分離純化原代大鼠EpSCs，無血清低鈣培養(yǎng)基為其提供營養(yǎng)。 2．采用免疫化學技術：細胞免疫熒光（Immunofluorescence，IF）、蛋白印記法（Western Blotting，WB）、流式細胞術（Flow Cytometric，F(xiàn)CM）檢測EpSCs表面標記物β1-整合素、α6-整合素、角蛋白19（Cytokeratin19，K19）及CD71的表達情況，確定其是否符合EpSCs標準。 3．通過IF單染和雙染法檢測大鼠EpSCs中α-MSH及其受體MC1R的表達情況，并采用逆轉錄聚合酶鏈式反應（Reverse transcription polymerase chainReaction，RT-PCR）檢測受體MC1R的表達情況。 結果： 1．倒置相差顯微鏡下觀察，細胞培養(yǎng)2-3天，即有小克隆形成，并呈不規(guī)則橢圓形；培養(yǎng)4-5天，細胞增殖明顯加快，克隆變大；培養(yǎng)7-8天，細胞融合至70-80%，形成鋪路石狀，折光性好。 2．檢測大鼠EpSCs的表面標記物發(fā)現(xiàn)：WB檢測培養(yǎng)的細胞表達β1-整合素；共聚焦顯微鏡下觀察，細胞可同時表達K19和β1；FCM檢測結果顯示，2代EpSCs高表達α6，，低表達CD71。 3．IF檢測到EpSCs表達α-MSH；免疫熒光雙標法發(fā)現(xiàn)，K19與MC1R、α6與MC1R表達均呈陽性；RT-PCR顯示體外培養(yǎng)的EpSCs表達受體MC1R及α-MSH的前體及合成酶。 結論：“胰酶兩步消化+IV型膠原差速貼壁法”可獲得形態(tài)均一、活力好、純度較高的大鼠EpSCs；體外培養(yǎng)的大鼠表皮干細胞表達α-MSH及受體MC1R。
[Abstract]:Research background:
Epidermal stem cells (EpSCs, Epidermal stem cells) since its discovery has been the focus of research, as the specific stem cells in skin tissues, with a high degree of self-renewal and multilineage differentiation potential, which not only maintain the skin and skin daily The new supersedes the old. environment in the steady state, and is closely connected with the wound repair. When subjected to external injury stimulation, EpSCs can actively participate in skin damage repair, through de adhesion, migration, proliferation, and epidermal terminal cell differentiation induced by epidermal keratinocytes to wound and promote wound coverage, crawling, re epithelialization, and ultimately achieve the purpose of wound repair.
Alpha melanocyte stimulating hormone (alpha -MSH, alpha -melanocyte stimulating hormone) was originally discovered because it is in the promotion of melanogenesis, regulate skin pigmentation effect. Recent studies have proved that the alpha -MSH in anti-inflammatory, antipyretic, antibacterial, nerve nutrition, energy balance, regulating cardiovascular function, biological behavior influence animal and maintain relatively stable immune function has played an important role in the regulation of the melanocortin receptor alpha -MSH. (melanocortin receptor, MCR) combination with its physiological function of.MCR is a G protein coupled receptor, truseal has 5 kinds of MCR have been cloned and qualitative, found in the skin is mainly MC1R (Melanocortin1receptor, MC1R), MC2R (Melanocortin2receptor, MC2R) and MC5R (Melanocortin5receptor, MC5R). MC1R has been found in melanocytes, keratinocytes, sebaceous gland cells, fibroblast cells, Langerhans cells, dermal free Plague and endothelial cells are expressed in almost all types of skin tissue cells, and the most closely related to these cells.
In this experiment, EpSCs isolated from rat skin basal layer, and identification; simultaneous detection of EpSCs alpha -MSH and its receptor MC1R expression, to lay the foundation for the study of alpha -MSH's effects on EpSCs and other follow-up experiments, in order to further enrich and perfect the theory basis of wound healing and clinical application.
Method錛

本文編號：1577979