A New Model For Long-term Observation Of In Vivo Functions Of Human Melanocytes

ObjectivesMelanocytes are important functional cells in the body,which can protect the skin from the damage of ultraviolet ray.Future research on the characteristics of melanocytes will help to understand the pathogenesis of pigment disorders and develop new treatment strategies.Compared with cell experiments,animal models are better for observing the in vivo functions of melanocytes.However,the existing animal transplantation model of melanocytes has some limitations,for example:abnormal localization of melanocytes after transplantation and short observation time.Therefore,a stable transplantation model is urgently needed to evaluate the biological characteristics and long-term function maintenance of human melanocytes in vivo.MethodsIn this study,we first transplanted the epidermal and dermal cells of neonatal mice into the back skin of immunodeficient mice by using silica gel chamber,establishing an improved hair follicle reconstruction model.Then,at different time points after transplantation,the number and proportion of hair follicles of at anagen,catagen and telogen were calculated to compare the hair cycle between the transplanted mice and normal mice.Finally,human melanocytes were mixed with the epidermal and dermal cells of neonatal mice for co-transplantation,and anti-asialo GM1 was injected to suppress immune rejection.Observe the integration site,function and survival time of human melanocyte in immunodeficient mice through Masson-Fontana staining,Dopa staining and immunofluorescence staining.Results1.Through the modified chamber model,5×10~6 epidermal cells and 5×10~6 dermal cells of C57BL/6 neonatal mice were transplanted into the back of nude mice,and the hair follicles could be successfully reconstructed.The wound scabbed and healed at 2 weeks,hair begin grows at 3 weeks.The length and density increased gradually.According to the observation from 2 weeks to 8 weeks after transplantation,the hair density of reconstructed hair follicles was close to that of normal C57BL/6 mice,with no significant statistical difference.2.The proportion of hair follicles in anagen,catagen and telogen also showed no significant difference,and the hair cycle was basically synchronized.At 3 weeks of co-transplantation with human epidermal melanocytes,Masson-Fontana positive and Dopa positive cells were found in the reconstructed hair bulb and hair shafts,indicating that these human epidermal melanocytes could integrate into the physiological distribution area and function.We also found human-specific nuclei hNuclei and melanocyte markers TYRP1 co-positive cells in the hair follicles by immunofluorescence staining,which indicate the melanocyte cells are human-derived.3.At 11 weeks after transplantation,Masson-Fontana staining and Dopa staining positive cells were still seen in the hair follicles,suggesting that these melanocytes could survive for a long time and function in immunodeficient mice.ConclusionsThus,this study established a mice model that can be used to observe the status of human melanocytes in vivo for a long time,which is helpful to further study the localization,migration,replication and differentiation of human melanocytes in vivo,and provide more references for the melanocyte transplantation in depigmentation diseases.