Experiment Study Of Dedifferentiation Of Human Epidermal Cells

ObjectiveSkin is the biggest organ of human body and it locates at body surface. It is likely to be injured. During the healing process, epidermal cells proliferate and migrate to cover the raw surface and then, differentiate. Epidermal cells develop a series of phenotype changing in the process of repairing. It is found that epidermal stem cells play a important role in the self-renewal and keeping stable structure and function of epidermis. It is very attracting to activate epidermal stem cells to promote the healing process of skin injury. Many researches find that it is the general phenomenon of activating stem cells or inducing cells dedifferentiating to accomplish endogenous regeneration. Dedifferentiated cells obtain the stem cell appearance and ability of proliferation and form new units to repair injury. In recent years, the study of dedifferentiation becomes a popular area in the research of wound repair. It is reported that epidermal cells could occur dedifferentiation during the healing of skin injury sometimes.Now, the means of studying epidermal cells mainly include three kinds of way. The first one is biopsy of human skin tissue. The second one is studying the cultured epidermal cells. The last one is investigating epidermal cells of animals to infer the mechanism of human epidermal cells.In this study, the first step is to create a proper animal model of human epidermal cells researching in vivo and then, to study the changing of their phenotypes. At last, we design a epidermic grafting animal model without basal layer of epidermis to investigate the dedifferentiation of epidermal cells.Methods1. Establishing animal model of epidermic graftingThe epidermis was isolated from human foreskin by digestion with protease. Then, it was grafted to the surface of the back of athymic mouse where the shin was resected or to the subcutaneous area without skin defect. So, two kinds of animal models were constructed. The epidermis would be taken in 3 days, 5 days and 7 days after grafting. Specimens were explored with HE staining and immunohistochemical staining (CK10, CK14, p63). The changes in phenotypes were observed.2. Exploring changes in phenotyes of epidermis in the second animal modelThe changes in phenotypes of xenografts were detected on the 3d, 5d and 7d with immunohistochemical method. The markers included CK10, CK14, CK19,β1-integrin, PCNA, p63 and c-Myc.3. Design animal model of epidermic grafting with eliminating basal layer of epidermis and study the dedifferentiation of epidermal cellsThe epidermis of human foreskin was isolated from dermis followed by digested with protease and the cells in stratum basale of the epidermis together with stem cells were eliminated by repeated adhesion to collagen typeⅣand flushing, then transplanted the treated epidermis to the subcutaneous area on the back of athymic BALB/c nude mice. The changes in phenotypes of the xenografts were detected on the 7d with immunohistochemical method and flow cytometry. In this study, the markers included CK10, CK14, CK19, PCNA, p63,α6-integrin and CD71 molecule.Results1. Epidermic graft survived at high percentage about 60% when transplanted to subcutaneous area, which was more than that to the back of full-thickness skin defect of athymic mouse. The epidermic cells of mouse would migrate to the inferior surface of graft and the grafts failed off at last if grafting to the back with defect of full-thickness skin. Survived epidermis proliferated, grew, and differentiated just like the changes of injured skin. The markers of CK14 and p63 were expressed in cells of the stratum basale.2. Cells of basal layer proliferated and expressed markers of p63, PCNA, CK14 when epidermic graft was transplanted to subcutaneous area of athymic mouse. Cells in spinous and granular layers express CK10. Cells expressing CK19 andβ1-integrin were more than the normal epidermis. Cells around basal layer expressed high level of c-Myc. The abnormal cells distributed in spinous and granular layers and expressed the same phenotypes with the cells of basal layer.3. The cells in stratum basale of the epidermis were eliminated by repeated adhesion to collagen typeⅣand flushing. Epidermal horn disappeared and no well-arranged basal layer cells and CK14 positive cells could be observed. Immunohistochemical examination of the survival xenografts showed that some cells were positive for both DAPI and either human CK19 orβ1 integrin in spinous and granular layers at day 7 after transplantation. These cells expressed p63 and PCNA. Furthermore, there was a significant increase in the percentage of bothα6~+CD71~- andα6~+CD71~+ populations in human epidermal sheet grafts after transplantation when compared with those before xenotransplantation (P＜0.05), as determined by two-color flow cytometry.Conclusions1. The animal model of human epidermis xenotransplantation can be successfully established by transplantation of epidermic graft to the subcutaneous area of athymic mouse. It provides a excellent model to research epidermal cells.2. Epidermal cells of xenotransplantation occur the same changes as that of injured skin with high expressing CK14, p63, PCNA. The more cells expressingβ1-integrin and CK19 suggest that xenotransplantation promote proliferation of epidermal stem cells.3. The abnormal cells distributed in spinous and granular layers and expressed the same phenotypes with transit amplifying cell and epidermal stem cell. It suggests that epidermal cells occur dedifferentiation to promote wound repair of skin in some conditions.4. The cells in stratum basale of the epidermis can be eliminated by repeated adhesion to collagen typeⅣand flushing.5. The dedifferentiation phenomenon of epidermal cells is approved by the study of animal model of epidermic grafting with eliminating basal layer of epidermis. In some conditions, differentiated epidermal cells could occur dedifferentiating to promote wound repair except for activating the silent epidermal stem cells.