| As the biggest organ of the body and one of tissues with the fastest self-renewal, the skin is the first line of defense against dehydration, injury and infection,and plays an important role in maintaining homeostasis and preventing invasion of microorganisms and chemical substances. Therefore, it is critically important to recover barrier function after skin injury. Conventional therapies for skin injury included autogenous skin transplantation, auto-skin graft, hetero-skin graft as well as artificial surrogate coverage. However, because of limited resources of donor skin, auto-skin graft and hetero-skin graft may result in high cost and have risks of immunological rejection or disease infection, which adds difficulties to clinical application. Therefore, how to promote repair of skin wound is now one of tough problems and studies on repair of skin wound and wound healing are of theoretical and practical significance.Adult stem cells (ASCs) are a kind of cell colony with potential of multi-directional differentiation. In the meantime, ASCs can be obtained from all kinds of organs and tissues, without involvement in ethics problem, and has wide prospect of clinical application, which contributes to the fact that ASCs have been one of research hot spots in recent years. At present, ASCs have been separated from most tissues and identified, which shows that ASCs can be induced to differentiate into cell types at same or different embryonic layers, indicating its potential of multi-directional differentiation. Several studies reported that ASCs especially BMSCs could obtain satisfactory results for repair of tissue injury.Wound is tissue injury in itself. Stem cells have potential of multi-directional differentiation, self-renewal and good proliferation and differentiation and can satisfy requirement of skin repair. Large number of experiments found that BMSCs could in vivo and in vitro differentiate into epidermal cells, fibroblasts and vascular endothelial cells, which provides theoretical basis for skin wound repair with stem cells.The aim of the study was to ascertain whether BMSCs participated in repair of skin wound by means of mobilization and recruitment and preliminarily explore mechanism of stem cells recruiting at wound site so as to cater fresh treatment methods for all kinds of skin defects.In the study, BMSCs were first separated and cultured and then were induced to differentiate and identified. Recruitment of the transplanted BMSCs in repair of skin wound was observed to make sure whether the transplanted BMSCs could recruit towards wound to facilitate repair of skin wound. Meanwhile, the possible mechanism was further explored. Based current chemotaxis theory, BMSCs were modified by means of gene transfection to promote their migration to the wound skin and further boost effect of BMSCs in repair of skin wound.Main results and conclusions are as follows:1.We used variability adherence method for separation and purification of BMSCs from male BALB/C mice and found that in vitro culture could obtain large quantity of amplification and passages, which contributes to a simple, stable and effective method for in vitor culture and amplification of BMSCs. Experimental assessment and investigation showed that in vitro BMSCs had potential of inducing osteogenesis and adipogenesis, which accorded with pacing factor of seed cells and could meet demand of further studies.2.Systemic irradiation with Co60 (irradiation dose at 3.5Gy) was used in this study to induce radiation damage of bone marrow in order to inactivate host bone marrow, which may keep space for transplanted BMSCs and reduce influence of autogeneic bone marrow stem cells. After transplantation of cultured BMSCs, a bone marrow transplantation chimera model was successfully established to simulate function of autogeneic BMSCs, which caters reference for establishment of combined injury model of mice with different immune function.3.The study involved three kinds of cell labeling methods, ie, CFDA-SE fluorescence labeling, Y chromosomal marker and BMSCs labeling with adenovirus vector carrying GFP gene. After the labeled BMSCs from male BALB/C mice were injected into female BALB/C mice via vena caudalis, fluorescence microscope was used detect the labeled BMSCs and real-time PCR were employed to detect expression of Y chromosome in wound tissues after histological section; DAPI counterstain confocal microscopy was used detect the labeled BMSCs in wound tissues after frozen section. The results could clearly proved that BMSCs could be recruited on the wound and participate in repair of skin wound.4.The wound healing time for BALB/C mice with normal immune function after injury was (14.00±1.41) days, while that for nude mice and SCID mice with immune defects were (17.16±1.17) days and (19.83±0.76) days respectively, with statistical difference compared with BALB/C mice (P<0.01).5.Fluorescent quantitation RT-PCR was used to detect gene expression of SDF-1 in wound tissues, which showed that gene expression of SDF-1 was elevated in BALB/C mice at day 1 after injury and reached peak at day 5 (P<0.01). Gene expression of SDF-1 was then gradually decreased (but still remained higher than control group) and reached similar level to control group on primary wound healing at day 14. Gene expression of SDF-1in skin wound of nude mice and SCID mice was gradually increased after injury and reached peak at day 7 after injury (P<0.01), which was delayed compared with BALB/C mice. Gene expression of SDF-1was then gradually decreased and maintained at higher level than control group before wound healing at day 14. The results indicated that SDF-1 is either important chemotatic factor regulating regional inflammation or key cytokine regulating repair of tissue or organ injury. SDF-1 is expressed in normal skins. After skin wound, expression of SDF-1 is increased, when BMSCs are recruited on the wound for repair of the wound. In addition, immune function of the organism will affect recruitment of BMSCs and result in late wound healing, which provides novel method for clinical treatment of different kinds of wounds and promotion of wound healing.6.We successfully constructed adenovirus expression vector Adv-CXCR4 of CXCR4. BMSCs were transfected and retransfused via vena caudalis to detect expression of chromosome Y in wound skin tissues, which showed that chromosome Y was detected in wound skin tissues at day 1 after injury either for BMSCs free from Adv-CXCR4 tranfection or transplanted BMSCs transfected with Adv-CXCR4. The expression peak emerged at day 5, which lasted for over 14 days. In the meantime, compared with BMSCs free from Adv-CXCR4 transfection, transplanted BMSCs transfected with Adv-CXCR4 could distribute on the wound of BALB/C mice, with statistical difference (P<0.01). After BMSCs transfected with Adv-CXCR4 were transplanted into BALB/C mice, the wound healing time were (12.46±1.17) days, with 1.5~2 days shorter than (14.00±1.41) days for skin wound treated with transplantation of BMSCs free from transfection with Adv-CXCR4.
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