PBX1 Promotes Proliferation Of Human Skin-derived Melanocytes And/or Melanocyte Stem Cells By ERK And The Preliminary Study On Its Mechanism

Background:Melanocytes are derived from neural crest.As the embryo develops,neural crest cells and their products form melanoblast through the dorsolateral and ventrolateral pathways,and migrate to the hair follicles,epidermis,uveal membrane,pia mater and inner ear.When the melanocyte lineage is under developmental disorders,it can cause loss of hearing and vision,skin pigmentation disorders,and melanoma.Environmental pollution and endotoxin can affect the development of melanocytes.The above diseases and toxicological evaluations are all studied with melanocytes and melanocyte stem cells,but they are limited in vitro and easy to differentiate,which can not meet the needs of cell biology research.PBX1 as a cellular transcription factor that maintains selfrenewal and pluripotency of stem cells,and promotes cell proliferation.Research purposes:To study the effect of overexpression of PBX1 on the proliferation of human skinderived melanocytes and/or melanocyte stem cells,and to study the related mechanism of overexpression of PBX1 on the proliferation of human skin-derived melanocytes and /or human skin melanocyte stem cells.Methods:1.Isolation,culture and identification of human skin-derived melanocytes and/or melanocyte stem cells.The foreskin tissues were obtained from adults and adolescents undergoing circumcision.They were cut to a size of 1 mm × 1 cm,immersed in Dispase II solution,and stored in a refrigerator at 4 ° C overnight to separate dermal and epidermal tissue.On the next day,the epidermal tissue were digested with 0.25% trypsin containing EDTA to acquier cells then cultured in a selective medium after centrifugation.The human-derived melanocytes specific biomarkers were detected by RT-PCR,immunofluorescence,and Western Blot;MTT assay the OD value and the proliferation curve of human skin-derived melanocytes and/or melanocyte stem cells were plotted,and the population doubling time was calculated.2.Preparation of overexpressed PBX1 human skin-derived melanocytes and/or melanocyte stem cellsThe psPAX2,pMD2 G and PBX1 plasmids were mixed with EndoFectinTM-MaX transfection reagent and transfected into 293 T cells.The virus supernatants were collected 36 h and 60 h after transfection,centrifuged and concentrated in an ultrafiltration tube,mixed with LV-Enhance,and added to human skin-derived melanocytes and/or melanocyte stem cells.After 3 days,the fluorescence expression of the cells was observed under a fluorescence microscope,the cells were counted,and the transduction efficiency was calculated.Immunofluorescence and Western Blot were used to detect the expression of overexpressed PBX1 human skin-derived melanocytes and/or melanocyte stem cells specific markers.3.Effect of overexpression of PBX1 on proliferation of human skin-derived melanocytes and/or melanocyte stem cellsMTT was used to detect the proliferation of overexpressed PBX1 human skinderived melanocytes and/or melanocyte stem cells;cell cycle was detected by flow cytometry and the proliferation index was calculated;Western Blot was used to detect the expression of key proteins STAT3,AKT and ERK in the proliferative signaling pathway.ERK inhibitors were administered,and their effects on the proliferation of human skin-derived melanocyte stem cells were detected by counting and Western Blot.The effect of the ERK inhibitor on human skin-derived melanocyte stem cells cycle was detected by flow cytometry.4.Mechanism of overexpression of PBX1 on proliferation of human skin-derived melanocytes and/or melanocyte stem cellsThe effect of overexpression of PBX1 on the expression of cycle-related proteins in human skin-derived melanocytes and/or melanocyte stem cells was detected by Western Blot.Results:1.Isolation,culture and identification of human skin-derived melanocytes and/or melanocyte stem cellsMicroscopically,it can be seen that the human skin-derived melanocytes isolated and cultured are in the form of two poles or three poles;the human skin-derived melanocyte stem cells are spindle-shaped.RT-PCR results showed that the cell obtained by adult foreskin tissue expression genes TYR,TYRP1,MITF,TYRP2,C-KIT,SOX10,PAX3;immunofluorescence and Western Blot showed that the cells expression protein TYR,TYRP1,MITF,which is human skin-derived melanocytes.RT-PCR results showed that cell obtained from adolescent foreskin tissue expression genes TYR,TYRP1,MITF,TYRP2,C-KIT,SOX10,PAX3,immunofluorescence,Western Blot detection,results can be seen: the cells expression protein MITF does not express TYR or TYRP1,which is human skin-derived melanocyte stem cells.The results of MTT were calculated: the doubling time of human skin-derived melanocytes was 141.2 h;the doubling time of human skin-derived melanocyte stem cell was 80.8 h.2.Preparation of overexpressed PBX1 human skin-derived melanocytes and/or melanocyte stem cellsMicroscopically,293 T cells transfected with PBX1 were adherently grown,in good condition,and the transfection efficiency was high.The collected virus is transduced into human skin-derived melanocytes and/or melanocyte stem cells.Fluorescence microscopy observation: With the prolongation of culture time,the percentage of fluorescence increased gradually,the cell refractive index was heigh,and the cell state was good.The Cytation3 cell imaging multi-microplate assay system positive rate of counting transduction,the results show that: human skin-derived melanocytes eGFP group positive rate was 94%,over-expressed PBX1 group positive rate was 82%.The positive rate of human skin-derived melanocyte stem cells eGFP group was 83%,and the positive rate of overexpressing PBX1 group was 71%.Immunofluorescence and Western Blot results showed that after overexpression of PBX1,human skin-derived melanocytes still expressed proteins TYR,TYRP1 and MITF.Human skin-derived melanocyte stem cells still express the protein MITF and do not express TYR or TYRP1.3.Effect of overexpression of PBX1 on proliferation of human skin-derived melanocytes and/or melanocyte stem cellsCompared with the corresponding eGFP group,the results of MTT showed that human skin-derived melanocytes and human/or melanocyte stem cells proliferated faster in the overexpressed PBX1 group;flow cytometry results showed that Overexpression of PBX1 promotes the increase of the proportion of S phase cells in human skin-derived melanocytes and/or melanocyte stem cells and increases the proliferation index;Western Blot results showed that Overexpression of PBX1 can increase the p-EKR of human skin-derived melanocytes and/or melanocyte stem cells;After the addition of the ERK inhibitor,the number of human skin-derived melanocyte stem cells overexpressing PBX1 was reduced,and the cell cycle progression was blocked.4.Effects of overexpressed PBX1 on human skin-derived melanocytes and/or melanocyte stem cell cycle-related proteins.Western Blot results showed that overexpression of PBX1 could down-regulate the expression of P21 and up-regulate the expression of E2 F and CyclinD1 in human skin-derived melanocytes.Overexpression of PBX1 could down-regulate the expression of P21 and up-regulate the expression of CyclinE1 and CDK2 in human skin-derived melanocyte stem cells.Conclusions:1.Successfully isolated and cultured human skin-derived melanocytes and /or human skin-derived melanocyte stem cells in vitro.2.Overexpressed PBX1 human skin-derived melanocytes and/or melanocyte stem cells were successfully prepared.3.Overexpression of PBX1 promotes the proliferation of human skin-derived melanocytes and/or melanocyte stem cells by up-regulating phosphorylated ERK and affecting cell cycle-related proteins.