Establishment And Evaluation Of The Method For Isolation And Culture Of Mouse Pelage Dermal Papilla Cells

Background:With the development of tissue engineering and regenerative medicine,reconstruction of tissue engineering hair follicle will be a promising solution for alopecia treatment.DPCs are important seed cells in the process of hair follicle reconstruction.But,DPCs gradually lose hair-inductive capacity during subculture.Many strategies are used to maintain or restore the inductive ability of DPCs in vitro,.However,it is still difficult to obtain a sufficient number of DPCs with inductive ability in a short time.At present,the reconstruction of tissue engineering hair follicle is in the experimental stage,DPCs are usually derived from mouse vibrissa hair follicle or surgical disposal of human scalp hair follicle,the source is very limited.Therefore,we tried to isolate and culture mouse pelage DPCs in order to provide a new source of DPCs.Objective:(1)To isolate and culture pelage DPCs of C57 mice.(2)To detect the hair-inductive ability of cultured DPCs in vitro.(3)To investigate the feasibility of using cultured pelage DPCs of mice for hair follicle reconstruction.Methods:(1)Isolation and culture pelage DPCs of C57 miceDorsal skin of C57BL/6J mice at 5w was removed.After digestion and Ficoll gradient centrifugation,DPs were obtained.The appearance,attachment and the migration of the DPs were observed under the microscope.The adherence rate of the DPs was calculated and cell growth was observed.(2)Detection the hair-inductive ability of pelage DPCsMice pelage DPCs at passage 3,mice vibrissa DPCs at passsage 3 and mice dermal fibroblast were performed to detect the biological characteristics that related to hair follicle inducing ability.qRT-PCR was conducted to evaluate the gene expression of ALP,(3-catenin and Versican.Immunofluorescence staining was performed to qualitatively detect the protein expression of ALP,?-catenin and Versican.(3)Cultured pelage DPCs were used to reconstruct hair follicleMice pelage DPCs at passage 3,mice vibrissa DPCs at passsage 3 and mice dermal fibroblast were engrafted with neonatal mice epidermis respectively to reconstruct hair follicle.Observe hair follicle formation,development and growth of the hair shaft.4weeks after transplantation,the skin at the transplantation site was harvested for paraffin section and HE staining.Result(1)Isolation and culture pelage DPCs of C57 mice Freshly isolated pelage DPs were round or oval and the adherence rate was 90%.Subcultured DPCs show a long spindle-like fiber-like arrangement and swirl-like growth.As the number of passages increased,the dermal papilla cells gradually lost their aggreative growth ability.(2)Detection the hair-inductive ability of pelage DPCsqRT-PCR,immunofluorescence test showed that:signature genes and proteins of DPCs including ALP,?-catenin and Versican were expressed in mouse pelage DPCs.Expression of these distinct DPC markers associated with hair follicle-inductivity were all expressed in mouse vibrissa DPCs.However,no significant expression was found in fibroblasts.(3)Cultured pelage DPCs were used to reconstruct hair follicleNo regenerated hair growth on the skin was observed when fresh epidermal cells were transplanted only.When fresh epidermal cells co-grafted with fibroblasts,histological examination revealed that no regenerated hair follicles were present in the grafted site.When fresh epidermal cells co-grafted with pelage DPCs or fresh epidermal cells co-grafted with vibrissa DPCs,hair follicle structures were observed.Conclusion(1)Enzyme digestion combined with Ficoll gradient centrifugation could be used to obtain mice pelage dermal papilla.This method has the advantages of high yield,high separation efficiency,low labor intensity and high adherence rate.Besides,it does not significantly affect the cell morphology and growth pattern of DPC in primary culture in vitro.(2)Results from qRT-PCR,immunofluorescence staining demonstrated that:cultured mice pelage DPCs can express hair follicle-inductive markers including ALP,?-catenin and Versican.There is no significant difference of markers expression between mice pelage DPCs and vibrissa DPCs.(3)Cultured mice pelage DPCs posses the ability to induce hair follicle regeneration and can be used for hair follicle reconstruction experimental study.