Effects Of Long-term Trypsinization On Human Epidermal Melanocytes

Objective:To explore the biological characteristics including morphology,survival rate,proliferative rate,melanin content,expressions of immature and mature markers of human epidermal melanocytes after being treated with long-term trypsinization(LTT),and to invest the underlying possible mechanisms.Methods:The normal foreskin was obtained from the circumcision,and after a series of operations,including specimen trimming,enzymatic digestion,primary culture of the epidermis and differential trypsinization,the adherently growing epidermal melanocytes were obtained.These cells were divided into two groups and treated with LTT(2 h)or routine trypsinization(10 min),respectively.After the survival rates of the two groups of cells were evaluated by trypan blue staining immediately,they were seeded into culture plates.After 24 hours incubation,the cells seeded in 24-well culture plates underwent detection about autophagy and immunofluorescence stainings with antibodies against MCs markers,including HMB45,TYR and Nestin.The respective expressions of TYR,Nestin,c-Kit and MITF mRNA were determined by reverse transcription-polymerase chain reaction(RT-PCR)when the cells in 6-well plates reached approximate 90%.The proliferative rates of cells in the 96-well plates were detected by CCK-8 method on the 1st,3rd,5th and 7th day of cell incubation respectively.Their melanin contents were detected on the second day.Results:After treatment of LTT,more small and bipolar MCs appeared in the experimental group.Cell viability test showed that most MCs survived treatment of LTT.There was no significant difference of cell survival rates between the LTT and control groups.The cell proliferative rates and melanin content in the LTT group were lower than those in the control group,respectively.The increased autophagy was detected after LTT.The results of immunofluorescence staining demonstrated that TYR expression was reduced in the LTT group,HMB45 expression was not different between the two groups,Nestin expression was increased slightly in LTT group.The results of RT-PCR suggested that compared with the control group,the levels of mRNA encoding TYR and MITF were lower,while Nestin mRNA was higher in LTT group.Conclusions:Human epidermal MCs could tolerate LTT and further dedifferentiate in vitro.The mechanism may be partly related to autophagy.Immature melanocytes obtained by the LTT may be a potential source of cells for melanocyte transplantation therapy.