Effect Of Luteolin-7-glucoside On Proliferation Of Human Epidermal Stem Cells And Its Mechanism

Epidermal stem cells(EpSCs)play a vital role in epidermal regeneration,skin homeostasis and wound healing.EpSCs possess self-renewal capacity and proliferative potential,and are ideal seed cells for fabrication of tissue engineering skin.The search for compounds that promote epidermal cell proliferation will be helpful for getting more seed cells for preparation of engineering skin tissue in vitro.Luteolin-7-glucoside(L7G)has been reported to promote skin wound healing through inhibition of inflammation oxidative stress and hyaluronidase/collagenase activity.ObjectiveTo investigate the effect of L7G on proliferation of human EpSCs and explore the underlying mechanisms.Methods1.Epidermal cell suspension was prepared by sequential digestion of human skin tissues with dispase and trypsin,and the EpSCs were isolated by their rapid adherence to culture plates coated with type IV collagen.EpSCs were identified by detecting EpSCs markers through immunofluorescence staining and flow cytometry assay.2.The effect of L7G on proliferation of EpSCs was examined by MTT,BrdU incorporation assay and Ki67 immunofluorescence staining.The effect of L7G on EpSCs cell cycle phase distribution was analyzed by flow cytometry.The effect of L7G on migration of EpSCs was investigated by an in vitro scratch healing model.3.The expression of EpSCs proliferation-related genes was examined by RT-PCR and Western blot.4.Human skin tissues were cultured with L7G in vitro and examined the thickness of epidermis and the number of EpSCs by HE staining,immunohistochemical and immunofluorescence staining of EpSC markers.Results1.EpSCs isolated from human skin showed cobblestone-like morphology in culture.Immunofluorescence staining showed that more than 90%of the cells were CK19 and ?1 integrin-positive.Flow cytometry assay showed that 92.87%of the cells expressed high level of a6 integrin and low level of CD71.These results demonstrated the successful isolation and culture of skin EpSCs.2.L7G at 0.1-1 ?M can promote EpSCs proliferation in a dose-dependent manner.The pro-proliferative effect of L7G on EpSCs was confirmed by BrdU incorporation assay and Ki67 immunofluorescence staining.Cell cycle analysis showed that treatment of EpSCs with L7G decreased the cell number in the G1 phase and increased the cell number in S phase.1 ?M L7G significantly enhanced EpSCs migration.3.Mechanistic studies showed that L7G significantly induced the expression of ?-catenin,c-Myc and cyclin D1,A2,E1.L7G promoted EpSCs proliferation through upregulating?-catenin and c-Myc.4.Incubation of human skin tissue explants with L7G significantly increased the thickness of epidermis and increased the number of a6 integrin-positive and ?1 integrin-positive cells at the basal layer of the epidermis.In addition,the expression of p63 and the cell proliferation antigen were significantly enhanced by L7G in epidermal cells.These results indicate that L7G can promote EpSCs proliferation in vitro and in skin tissue explants.ConclusionsL7G promotes the proliferation of the EpSCs in vitro by upregulating the expression of?-catenin,c-Myc and cyclins.L7G promotes EpSC proliferation and increases the thickness of epidermis in cultured human skin tissue.