本文選題：表皮　切入點(diǎn)：干細(xì)胞　出處：《南華大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景： 表皮干細(xì)胞（EpSCs，Epidermal stem cells）自發(fā)現(xiàn)以來(lái)一直是研究的熱點(diǎn)，作為皮膚組織的特異性干細(xì)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(nèi)環(huán)境穩(wěn)態(tài)，而且與創(chuàng)面修復(fù)緊密相連。當(dāng)受到外界損傷刺激時(shí)，EpSCs可主動(dòng)參與損傷皮膚的修復(fù)，通過(guò)脫粘附、遷移、增殖，并向表皮終末細(xì)胞分化，誘導(dǎo)表皮角質(zhì)形成細(xì)胞向創(chuàng)面爬行，進(jìn)而覆蓋創(chuàng)面，促進(jìn)再上皮化，最終達(dá)到修復(fù)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(jìn)黑素生成、調(diào)節(jié)皮膚色素沉著方面的作用。近年來(lái)大量研究證實(shí)，α-MSH在抗炎、退熱、抗菌、營(yíng)養(yǎng)神經(jīng)、能量平衡、調(diào)節(jié)心血管功能、影響動(dòng)物生物學(xué)行為及維持免疫功能相對(duì)穩(wěn)定等方面都發(fā)揮了重要的調(diào)控作用。α-MSH通過(guò)與黑皮素受體（melanocortin receptor，MCR）結(jié)合發(fā)揮其生理功能。MCR是一個(gè)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(xì)胞、角質(zhì)形成細(xì)胞、皮脂腺細(xì)胞、成纖維細(xì)胞、朗格漢斯細(xì)胞、真皮免疫細(xì)胞和內(nèi)皮細(xì)胞等幾乎所有類(lèi)型的皮膚組織細(xì)胞中均有表達(dá)，且與這些細(xì)胞的關(guān)系最為密切。 本實(shí)驗(yàn)從大鼠皮膚基底層分離培養(yǎng)出EpSCs，并進(jìn)行鑒定；同時(shí)檢測(cè)EpSCs中α-MSH及其受體MC1R的表達(dá)情況，為研究α-MSH對(duì)EpSCs的調(diào)控作用等后續(xù)實(shí)驗(yàn)打下基礎(chǔ)，以便進(jìn)一步豐富完善創(chuàng)面修復(fù)的基礎(chǔ)理論和臨床應(yīng)用。 方法： 1．實(shí)驗(yàn)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(wú)血清低鈣培養(yǎng)基為其提供營(yíng)養(yǎng)。 2．采用免疫化學(xué)技術(shù)：細(xì)胞免疫熒光（Immunofluorescence，IF）、蛋白印記法（Western Blotting，WB）、流式細(xì)胞術(shù)（Flow Cytometric，F(xiàn)CM）檢測(cè)EpSCs表面標(biāo)記物β1-整合素、α6-整合素、角蛋白19（Cytokeratin19，K19）及CD71的表達(dá)情況，確定其是否符合EpSCs標(biāo)準(zhǔn)。 3．通過(guò)IF單染和雙染法檢測(cè)大鼠EpSCs中α-MSH及其受體MC1R的表達(dá)情況，并采用逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)（Reverse transcription polymerase chainReaction，RT-PCR）檢測(cè)受體MC1R的表達(dá)情況。 結(jié)果： 1．倒置相差顯微鏡下觀察，細(xì)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(xì)胞增殖明顯加快，克隆變大；培養(yǎng)7-8天，細(xì)胞融合至70-80%，形成鋪路石狀，折光性好。 2．檢測(cè)大鼠EpSCs的表面標(biāo)記物發(fā)現(xiàn)：WB檢測(cè)培養(yǎng)的細(xì)胞表達(dá)β1-整合素；共聚焦顯微鏡下觀察，細(xì)胞可同時(shí)表達(dá)K19和β1；FCM檢測(cè)結(jié)果顯示，2代EpSCs高表達(dá)α6，，低表達(dá)CD71。 3．IF檢測(cè)到EpSCs表達(dá)α-MSH；免疫熒光雙標(biāo)法發(fā)現(xiàn)，K19與MC1R、α6與MC1R表達(dá)均呈陽(yáng)性；RT-PCR顯示體外培養(yǎng)的EpSCs表達(dá)受體MC1R及α-MSH的前體及合成酶。 結(jié)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(xì)胞表達(dá)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.
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本文編號(hào)：1577979