Effect of δ-opioid receptor on bioactivity of human epidermal stem cells in vitro

Abstract

Objective To observe the effect of δ-opioid receptor on proliferation and migration of human epidermal stem cells (hESCs) in vitro so as to offer treatment theory for skin injury.Methods hESCs from fresh foreskin tissues of normal young volunteers were isolated and cultured by enzyme digestion and differential adherence technique.Immunofluorescent staining was used to determine expression of integrin β1 and cytokeratin 19 (CK19) and flow cytometry was used for cell count.Second generation of cells were cultured for 5 consecutive days with keratinocyte serum-free medium (K-SFM) supplemented with 1 nmol/L (D-Ala2,K-Leu5)-enkephalin in Group A,with K-SFM supplemented with 1 nmol/L naltrindole and 1 nmol/L (D-Ala2,K-Leu5)-enkephalin in Group B,and with isolated K-SFM in Group C.Cellular division and proliferation were detected by MTT method.An in vitro 100 μm scratch-wound model was created on the confluent monolayer cells at 24 hours of incubation.Cells migrating from the wound margin were determined by inverted phase contrast microscope at 24,48,72,and 96 hours after wound formation,while wound closure rate was calculated at 72 hours.Results Primary cultured hESCs presented cobblestone-like shape after adherence growth,Immunofluorescence staining showed positive results for integrin β1 and CK19 and cell purity reached 95.6％.Moreover,MTT findings revealed proliferation of hESCs enhanced significantly in Group A,but lowered in Group B as compared to Group C (P ＜ 0.05).hESCs migrated from the wound margin in all groups at 24 hours.However,more migrated cells were seen in Group A than in Group C and less in Group B than in Group C.Rate of wound closure was (89.5 ±0.7)％ in Group A,(76.1 ±0.3)％ in Group B,and (81.1 ±0.6)％ in Group C at 72 hours,indicating significant differences among groups (P ＜ 0.05).Conclusion Activation of δ-opioid receptor promotes the proliferation and migration of hESCs in vitro and may be implicated in wound healing. Key words: Epidermis ; Stem cells ; Receptors, opioid ; Cell proliferation