大鼠表皮干细胞体外培养和α-MSH及MC1R表达研究

发布时间：2018-03-07 04:32

  本文选题：表皮　切入点：干细胞　出处：《南华大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景： 表皮干细胞（EpSCs，Epidermal stem cells）自发现以来一直是研究的热点，作为皮肤组织的特异性干细胞，具有高度的自我更新能力及多向分化潜能，其不仅维持皮肤日常的新陈代谢及皮肤内环境稳态，而且与创面修复紧密相连。当受到外界损伤刺激时，EpSCs可主动参与损伤皮肤的修复，通过脱粘附、迁移、增殖，并向表皮终末细胞分化，诱导表皮角质形成细胞向创面爬行，进而覆盖创面，促进再上皮化，最终达到修复创面的目的。 α-促黑素（α-MSH，α-melanocyte stimulating hormone）最初被人们发现是因其在促进黑素生成、调节皮肤色素沉着方面的作用。近年来大量研究证实，α-MSH在抗炎、退热、抗菌、营养神经、能量平衡、调节心血管功能、影响动物生物学行为及维持免疫功能相对稳定等方面都发挥了重要的调控作用。α-MSH通过与黑皮素受体（melanocortin receptor，MCR）结合发挥其生理功能。MCR是一个G蛋白偶联型受体，至今已有5种MCR被克隆和定性，皮肤中发现的主要是MC1R（Melanocortin1receptor，MC1R）、MC2R（Melanocortin2receptor，MC2R）及MC5R（Melanocortin5receptor，MC5R）。已发现MC1R在黑素细胞、角质形成细胞、皮脂腺细胞、成纤维细胞、朗格汉斯细胞、真皮免疫细胞和内皮细胞等几乎所有类型的皮肤组织细胞中均有表达，且与这些细胞的关系最为密切。 本实验从大鼠皮肤基底层分离培养出EpSCs，并进行鉴定；同时检测EpSCs中α-MSH及其受体MC1R的表达情况，为研究α-MSH对EpSCs的调控作用等后续实验打下基础，以便进一步丰富完善创面修复的基础理论和临床应用。 方法： 1．实验以新生SD大鼠背部皮肤为材料，采用“胰酶两步消化+IV型胶原差速贴壁法”分离纯化原代大鼠EpSCs，无血清低钙培养基为其提供营养。 2．采用免疫化学技术：细胞免疫荧光（Immunofluorescence，IF）、蛋白印记法（Western Blotting，WB）、流式细胞术（Flow Cytometric，FCM）检测EpSCs表面标记物β1-整合素、α6-整合素、角蛋白19（Cytokeratin19，K19）及CD71的表达情况，确定其是否符合EpSCs标准。 3．通过IF单染和双染法检测大鼠EpSCs中α-MSH及其受体MC1R的表达情况，并采用逆转录聚合酶链式反应（Reverse transcription polymerase chainReaction，RT-PCR）检测受体MC1R的表达情况。 结果： 1．倒置相差显微镜下观察，细胞培养2-3天，即有小克隆形成，并呈不规则椭圆形；培养4-5天，细胞增殖明显加快，克隆变大；培养7-8天，细胞融合至70-80%，形成铺路石状，折光性好。 2．检测大鼠EpSCs的表面标记物发现：WB检测培养的细胞表达β1-整合素；共聚焦显微镜下观察，细胞可同时表达K19和β1；FCM检测结果显示，2代EpSCs高表达α6，，低表达CD71。 3．IF检测到EpSCs表达α-MSH；免疫荧光双标法发现，K19与MC1R、α6与MC1R表达均呈阳性；RT-PCR显示体外培养的EpSCs表达受体MC1R及α-MSH的前体及合成酶。 结论：“胰酶两步消化+IV型胶原差速贴壁法”可获得形态均一、活力好、纯度较高的大鼠EpSCs；体外培养的大鼠表皮干细胞表达α-MSH及受体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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