Rat Epidermal Stem Cells Cultured In Vitro And Expression Of α-MSH And MC1R

Background:Epidermal stem cells (EpSCs, Epidermal stem cells) since its discovery has havebeen a research focus, as the skin tissue-specific stem cells have a high degree ofself-renewal and differentiation potential, which not only maintain the skin’s dailymetabolism and the skin environmental stability state, but also is closely linked withthe skin wound repair. When subjected to external damage stimuli, EpSCs activelyget involved in the repair of damaged skin, through the deadhesion, migration,proliferation and differentiation of epidermal terminal cells, inducingepidermalkeratinocytes to wounds crawling, and then covering the wound and promotingwound re-epithelialization, EpSCs ultimately achieve the purpose of wound repair.α-melanocyte stimulating hormone (α-MSH, α-melanocyte stimulating hormone)was discovered initially because of its promoting melanogenesis, and its regulation ofskin pigmentation area. In recent years, a large number of studies have confirmed,α-MSH plays an important role in the anti-inflammatory, antipyretic, anti-bacterial,nerve nutrition, energy balance, regulating cardiovascular function, the biologicaleffects of animal behavior and maintaining relative stability and other aspects ofimmune function. α-MSH combined with the melanocortin receptor (melanocortinreceptor, MCR) plays its physiological functions. MCR is a G protein-coupledreceptors, with five kinds of MCRs cloned and characterized. What is mainlydiscovered in the skin, are MC1R (Melanocortin1receptor, MC1R), MC2R(Melanocortin2receptor, MC2R) and MC5R (Melanocortin5receptor, MC5R).MC1R which has already been found is expressed in melanocytes, keratinocytes,sebaceous gland cells of almost all types, fibroblasts, Langerhans cells, dermal cellsand other immune cells and endothelial cells of the skin tissues, and these cells themost closely related. During this research, we isolate and culture EpSCs from rat basal layer of theskin, and is later identified; meanwhile, we simultaneously detect the expression ofα-MSH and its receptor MC1R in EpSCs, which lay the foundation in the study ofother follow on EpSCs experiments such as the regulation of α-MSH, and also in thestudy of further improving the other follow on EpSCs experiments of wound repair.Methods:1. An experiment with the dorsal skin of newborn SD rats materials, the“two-step trypsin digestion+IV collagen differential adherence Law” isolated andpurified primary rat epidermal stem cells in serum-free medium to provide nutrientscalcium.2. By immunocytochemistry techniques: immunofluorescence (Immunofluores-cence, IF), Western blot method (Western Blotting, WB), flow cytometry (FlowCytometric, FCM) to detect cell surface markers:β1-integrin, α6-integrin, keratin19and CD71, to determine whether they meet the criteria of epidermal stem cells.3. By IF single and double staining dye test the expression of α-MSH and itsreceptor MC1R in rat EpSCs, and reverse transcription polymerase chain reaction(Reverse transcription polymerase chain Reaction, RT-PCR) to detect receptor MC1Rexpression.Results:1. It is observed that cell cultured for2-3days, in the inverted phase contrastmicroscope a small colony is formed and shaped in irregular oval; cultured for4-5days, cell proliferation is significantly accelerated cloning larger; cultured for7-8days, the cells fuseto70-80%, the cobblestone-like, good refraction is formed.2. The results about cultured cells expressing surface markers β1-integrin, α6-integrin, keratin19and CD71showed that: The cultured cells were detectableexpression of β1-integrin for WB; This experiment cultured cells can express K19and β1simultaneously for laser scanning confocal microscope; The results show thathigh expression of α6, low expression of CD71of two generations EpSCs for flowcytometry (Flow Cytometric, FCM) detection. 3. The expression of α-MSH in EpSCs can be detected by IF; The expression ofK19and MC1R, α6and MC1R is discovered by immunofluorescence double labeling,which is positive; using RT-PCR technology determines the expression of MC1R invitro of EpSCs.Conclusions:Using “Trypsin digestion+IV collagen-step differential adhesion method”uniform morphology, good energy, high purity of rat EpSCs can be obtained; whetherthe expression of α-MSH and its receptor MC1R has a regulating effect on EpSCsremains yet to be proved.