Reparative And Regenerative Effects Of Stem Cells On Human Skin Tissue And The Key Regulatory Factors

Objective: To investigate the reparative and regenerative effects of human mesenchymal stem cells (MSCs) on skin and the key regulatory factors. On the one hand,we observed the reparative and regenerative effects of human MSC conditioned medium on photo-aged epidermal cells and discussed the effective secretion factors and their mechanism, for providing a new method for skin anti-aging and dedifferentiation. On the other hand, on the basis of previous study, we aimed to validate the key role of Pax6 gene on DNA methylation regulation in the process of sweat gland regeneration by dedifferentiated epidermal stem cells, and to evaluate the safety of sweat gland like (SGL)cells. Then further analyze the mechanism of sweat gland regeneration by the key regulatory factors.Methods: Iimmortalized human epidermal cells (HaCaT) were irradiated by quantitative ultraviolet to build the model of photo-aged epidermal cells. Human chorion-derived mesenchymal stem cells (CDSCs) were cultured and photo-aged epidermal cells were treated with different concentrations of CDSC conditioned medium(CDSC-CNM). The regenerative and reparative effects were observed through the changes of cell proliferation, ROS production and DNA damage determined by cell migration,fluorescence probe and comet assay experiments. Effective secreted factors were detected,and proteins of related pathway were determinated through the Western blot for analysis of the mechanism. Pax6 expression vector was constructed and dedifferentiated epidermal stem cells were cultured and divided into groups. The Pax6 plasmid was transfected into dedifferentiated epidermal stem cells and screened by G418 for the stable expression cell line. Demethylation agent (5-Aza-dC) was added for continue cultivation. The expression of mRNA of Pax6 was detected by Realtime-PCR. The sweat gland markers CEA and CK7 were detected by immunofluorescence assay. The regeneration effects of sweat glands and safety evaluation of sweat gland transplantation in foot of nude mice were verified.Results: The photo-aged epidermal cell model was successfully constructed. A certain concentration of human CDSC-CNM has repairative and regenerative effects on photo-aged epidermal cells, which can effectively enhance cell viability, reduce ROS production and alleviate DNA damage. It was found that EGF, TGF-?, IL-6 and IL-8 may be the effective secretory factors by the detection of CDSC-CNM. The factors may play a role through JAK/STAT and MAPK signaling pathway. The Pax6 plasmid was constructed and transfected into dedifferentiated epidermal stem cells. The positive clones were got by G418 screening. Realtime-PCR and Western blot showed the mRNA and proteins expression of CEA, CK7 in the cells of Pax6+5-Aza-dC group. Immunofluorescence staining indicated positive expression of CEA, CK7 proteins in the cells of Pax6+5-Aza-dC group. About 7/10 nude mice that had paw transplantation of Pax6+5-Aza-dC group cells showed positive results by iodine-starch sweating experiment.HE staining showed the sweat gland regeneration and basal layer sweat gland conduit connection in Pax6+5-Aza-dC group by histological examination, while no sweat gland regeneration was found in control groups. No tumor was found in the experimental groups after inoculated with SGL cells. But back tumors were found in control groups after inoculated with gastric cancer cells (MGC-803) three weeks. Histological sections showed no abnormal or tumor cells in the experimental groups, while a large number of irregular tumor cells in control groups.Conclusion: Human CDSC-CNM have regenerative and reparative effects on photo-aged epidermal cells. Cytokines such as b-FGF, EGF, TGF-, IL-6 and IL-8 would play a key role. The results provide a new method for the prevention and treatment of skin anti-aging, and provide the inducing conditions of epidermal cell dedifferentiation. Pax6 and 5-Aza-dC can promote dedifferentiated epidermal stem cells transfected into SGL cells and express the specific markers CEA, CK7 of sweat gland. The biological function of SGL cells in nude mice model had been verified and the safety of SGL cell transplantation had been proved, thus bringing hope to patients of large sweat gland injury for sweat gland regeneration clinically. Through discussing the mechanism of Pax6 methylation regulation,the theoretical system of sweat gland regeneration would be further improved.