| Burn injury is a kind of special trauma characterized by skin defect. Woundmanagement is the main challenge in burn therapy. How to cover and repair the wound asearly as possible is critical for burn surgeons. Previous studies have showed that thephysiological wound healing depend on the proliferation and differentiation of residualepidermal stem cells (ESCs), especially on ESCs located in the bulge of hair follicle.Usually, ESCs stay quiescencely in their specific niches, and can proliferate anddifferentiate to terminal cells to repair the wounds when ESCs initiate to migrate from theirniches under certain stimuli. Therefore, the migration of ESCs from their niche is one of thekey steps in the burn wound healing.In previous studies, both keratinocytes and macrophages had been proved to be able togenerate high level of Nitric Oxide (NO) in or around the burn wound during the woundhealing procedure. Especially, amount of NO was detected in the wound within the earlyphase after burn injury. Moreover, literatures showed that NO could enhance migration ofendothelial cell, broachi epidermal cell and other kinds of cells. Our preliminaryexperiments showed that NO enhance significantly the migration of HaCat cell in vitro. Thealteration of cytoskeleton, Rho GTPase and integrin beta1might be involved in theprocesses. Herein our study will throw light on the potential effects of NO on ESCsmigration and the underlying mechanisms.Objective: To explore the effect of nitric oxide (NO) on the adhesion、proliferation andmigration of epidermal stem cells in vitro and in vivo.Method:1. Human epidermal stem cells were isolated and cultured with the modified method of rapidadhesion to collagen Ⅳ. The harvested cells were identified by the expressions of epidermal stemcell markers, integrinβ1and keratin19, through immunofluorescence staining, Western Blotting andFlow Cytometry. In the presence of Nitric Oxide (NO) donor S-nitroso-N-acetylpenicillamine(SNAP) at different concentrations (0,1,10,100,500μmol/L), the effect of NO on the migration of cultured epidermal stem cells was detected by in vitro wound healing model and Transwell assay,respectively. After that, the effect of NO on the adhesion and proliferation of epidermal stem cellswas determined by CCK-8methods.2. Newborn mice were administered BrdU peritoneally to sign ESCs by retentionlabelling method.7weeks later, the mice were suffered with superficial partial-thicknessburns on their backs. L-arginine or L-NMMA was used to influence the production of NO.The BrdU-positive cell in the regenerated epidermis were detected and quantified byimmunohistochemistry staining at hour48after burn.Results:1. ESCs were enriched based on collagen type Ⅳ adhesiveness. Western blotting andimmunofluorescence staining showed that the rapid adherent cells were CK19and integrin β1positive. Flow Cytometric Analyses confirmed that in which around56.52%cells werea6briCD71dim-positive.2. ESCs migration was promoted in the presence of1~100μmol/L of SNAP in thescratch test model, and the higher migration speed of ESCs happened under the condition of100μmol/L SNAP.3. Transwell experiments showed that the number of cells transferred to a membrane inthe presence of100μmol/L of SNAP is significantly higher than without SNAP.4. The adhesion assay showed that cell absorbance values in presence of either100or500μmol/L SNAP were significantly higher than that in the presence of0μmol/L of SNAP.5、The proliferation of cultured cells was significantly increased in the presence of either100or500μmol/L of SNAP as compared to without SNAP.6. The superficial partial-thickness injury model was set up under the condition of65°C heating for3seconds by electrical scald instrument with constant temperature andpressure as histologically determined.7. Stem cell tracing experiment showed that injected BrdU could successfully label theproliferative cell nucleus, and the BrdU positive cells in skin were mainly located in theeminence of hair follicles after6-week of injection.8. In vivo experiments showed that the BrdU positive cells in the re-epithelized tissuesof L-arginine group and L-NMMA group were significantly different when compared withcontrol group. Conclusion:1. Exogenous NO could promote the migration and proliferation of human epidermal stem cellsin vitro；2. NO could significantly enhance the migration of ESCs after burn injury in vivo,which contributes to wound healing;... |
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