The Basic Research Of The Effects Of Cell Differentiated Regulation On The Repair And Regeneration Of Skin And Its Appendages' Injuries

Objective: To investigate the antigens expression of skin and its appendages, as well as the effects of cell differentiated regulation on the repair and regeneration of skin and its appendages' injuries.Methods: The antigens expression of skin and its appendages of fetus at different EGA and adult were detected by immunohistochemical method. MSCs and SGCs were isolated, cultured and expanded in vitro respectively. The antigens expression of MSCs and SGCs were detected by two-steps immunocytochemistry. MSCs were induced toward adipocytic lineage and osteogenic lineage differentiation, and were labeled with BrdU or DAPI. The confluence SGCs (2~4×10~5) were heat-shocked at 47 ℃ for 40 min and cooled for 1~2h at 37℃, then l~2×10~5 labeled human MSCs were added later and co-cultured with SGCs. The phenotype of human MSCs was detected, the percentage of phenotype conversion was calculated, and the factors and signaling pathways involved in the course were examined 7d after co-culture. The labeled rat MSCs were intravenously injected into allogeneic full-thickness skin wounds rats, then the labeled cells and the phenotype of these cells were detetected 3d, 14d and 28d after allogeneic transplantation. The epidermis of human foreskin was isolated from dermis followed by digested with protease, and the cells in stratum basale of the epidermis were eliminated by repeated adhesion to collagen type IV and flushing, then transplanted the treated epidermis onto the fresh full-thickness skin wounds about 1 cm in diameter on the back of athymic BALB/c nude mice, locally applied rhEGF and bFGF twice a day. The phenotype of the xenografts was detected 7d later.Results: Sweat glands expressed markers of CK7, CK8, CK14, CK18, CK19 and CEA. Hair follicles expressed CK7, CK14 and CK19. Sebaceous glandsexpressed CK8 and CK14. Basal layer expressed CK19 and CK14, while horny layer expressed CK10. Immunocytochemistry showed MSCs did not express the hematopoietic markers CD34 and CD45, as well as SGCs markers CK19 and CEA, while positively expressed CD29, CD44, CD71, CD 105 and CD 166. MSCs could be induced toward adipocytic lineage and osteogenic lineage differentiation. Both BrdU and DAPI stained nucleus of MSCs, and the percentage of BrdU labeling was about 74% and DAPI about 90%. SGCs were strongly positive for CK7, CK8, CK18, CK19 and CEA. After SGCs were heat-shocked at 47°C for 40 min, the majority of them remained adherent but many cells lost cell-cell contact as their cytoplasms retracted. When adult MSCs were cocultured with heat-shocked SGCs, a subset of adult MSCs differentiated into SGCs, and the percentage of differentiation was enhanced by EGF and injured microenviroment, while weakened by PD98059. ERK pathway, especially the pERK was involved in the phenotype conversion of human MSCs into human SGCs. There were labeled MSCs noted in the hair follicles, sebaceous glands and blood vessels in full-thickness wounds, and the incorporated cells in hair follicles and sebaceous glands were also positive for pan-cytokeratin. Immunohistochemical examination of the survival xenografts showed that some cells were positive for both DAPI and either human CK19 or p 1 integrin in spinous and granular layers at day 7 after transplantation. Furthermore, there was a significant increase in the percentage of both <535+CD71" and &6+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.Conclusions: Both adult bone marrow derived mesenchymal stem cells and dedifferentiation of the cells closed to the injuries involved in the wound healing of skin and its appendages.