The Study Of Human Umbilical Cord Mesenchymal Stem Cells Promote The Skin Wound Healing In Mice

Objective:To investigate the effect of topical human umbilical cord mesenchymal stem cells(hUC-MSCs)transplantation on wound healing after full-thickness excision dermal wounds in mice, and so as to provide theoretical basis for hUC-MSCs for treatment of skin wound repair.Methods:hUC-MSCs were isolated from the human umbilical cord tissues from a full-term neonate by tissue explants adherent method, and then cultured and identified with flow cytometry for cellular immune phenotypes. The obtained hUC-MSCs were induced in vitro to differentiation into osteoblasts and adipocytes. The CM-Dil fluorescent dye was used to tags h UC-MSCs, and to observe the biological characteristics of the marked cell, and to detect proliferation ability of change of CM-Dil labeled hUC-MSCs by MTT method. Using BALB/c mice to establish full-thickness excision skin wound model, and then mice were randomly divided into h UC-MSCs group and PBS group. The mice were injected of hUC-MSCs suspension and PBS fluid respectively around the wounds. The wound healing condition was examined after operation in 3, 7 and 14 d, and pathological changes were evaluated by HE staining. The expression of VEGF and CD34 were observed with immunohistochemical assay, the trace of transplanted hUC-MSCs in wound tissues and the cell proliferation of the wound were analyzed with immunofluorescence assay, the protein expression of VEGF was detected with Western blotting.Results:A large number of biological activities of hUC-MSCs were obtained by tissue explants adherent method. Flow cytometry results showed that the cultured cell was h UC-MSCs, and the obtained cells could be induced to osteogenic and adipogenic cells after differentiation. CM-Dil fluorescent dyes can mark hUC-MSCs effectively, and the positive percentage of marker was(95.90 ± 1.79), continuous subculture to 15 d the positive percentage of marker was(87.90 ± 1.90). MTT method results showed that no influence to the hUC-MSCs proliferation ability by CM-Dil fluorescent dyes. The wound healing rate was obviously higher at all time points in the hUC-MSCs group(P<0.05). HE staining indicated that the wound healing quality is better in the former than the latter group. Immunohistochemical display that the expression of VEGF reached the highest in wound at 3 d, and then reduced gradually at 7 and 14 d in the hUC-MSCs group, but still higher than in the PBS group. The number of vessels in the wound of hUC-MSCs group were significantly higher than that of the PBS group at 3 and 7 d(P < 0.05), but there was no significant difference at 14 d(P > 0.05). Immunofluorescence show that CM-Dil positive cells slightly decreased in 3 and 7 d(P > 0.05), but reduced significantly at 14 d(P < 0.05). The percentage of BrdU positive cells in hUC-MSCs group in new epidermis were higher than that in the PBS group at 7 and 14 d(P < 0.05), in granulation tissue of wound the percentage of BrdU positive cells was increased to PBS group in 3 and 7 d(P < 0.05), while there was no significant difference in two groups in 14 d(P > 0.05). Western blotting assay showed that the protein expression level of VEGF was obviously higher than that in hUC-MSCs group than in the PBS group after operation at 3, 7, and 14 d(P < 0.05).Conclusion: h UC-MSCs transplantation applied can survive in the host, and by enhancing the expression of VEGF, promote angiogenesis and cell proliferation and accelerate skin wound healing of wound repair.