| Skin tissue has high ability of regeneration. Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish the worn out or damaged cells in tissue during life. The balance between cell division and cell loss is essential for epidermal homeostasis and must be maintained for life. With the developments of molecular biology,cell biology,tissue engineering and biotechnology, epidermal stem cells(ESCs) are become more and more important in gene therapy and cell therapy because of their distinguished biological characters. How to isolate and culture ESCs is of great importance not only for the study on tissue repair in trauma but also on skin cancer.Many cells and cytokines are involved in the complex process of epidermal wound healing. At the early stage, a great deal of exudate was generated in wound margin. Variety of cytokines play key roles in wound healing process, mainly including regulating various cell reactions, releasing of extracellular matrix, as well as affecting cell proliferation, migration and synthesisSDF-1 was secreted by the stromal cells and could mediate adult stem cells migration toward damaged region in a variety of adult tissue injuries and repair processes. Therefore, we speculated that SDF-1 might be expressed around wound, and had effect on the distribution and number of ESC in this area.Objectives:1. Epidermal stem cells (ESCs) were isolated from foreskins,separated based on collagen type IV adhesiveness, and cultured in serum free medium in vitro. Their growth was observed so we can establish an ideal method of ESC sorting and culture.2. To observe the effects of stromal cell derived factor-1(SDF-1) on ESCs proliferation and migration, and to explore its mechanism.3. To build a three-dimensional equivalent of full thickness skin wound model, observe SDF-1 expression in the process of skin wound healing and then analyze their correlation. Method:1. ESCs were isolated from human foreskins by dispase II-trypsin combined digestion. the culture dishes were coated with human placenta collagen typeⅣ, then ESCs were planted on human placenta collagen typeⅣ-coated dishes and cultured in Defined Keratinocyte Serum Free Medium(DK-SFM). The change on ESCs morphology, colony forming efficiency and the expression of surface markers (CD71,α6-integrin,β1-integrin, CK19 and PCNA) were detected.2. ESCs proliferation under the effect of different concentration of SDF-1 or AMD3100 was measured by MTT assay. ESCs migration and chemotaxis under the effect of different concentration of SDF-1 were measured by wound scrap experiment.3. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a rat tail collagen matrix, were grown on cell culture dishes. After 72 hour, primary human keratinocytes were seeded on this base. One week later, an air-lift transition was performed, leading to the differentiation of the ESCs, which were macroscopically visible as "three-dimensional skin equivalents" after a couple of days. We then used liquid nitrogen freezing metal rod to establish a full-thickness frostbite wounds in vitro. The distribution of SDF-1 on the wound margin, as well as the distribution of ESC under the effect of SDF-1 solely or combined with AMD3100, were detected by immunohistochemical staining on 3rd, 7th, and 10th day of frostbite,.Result:1. ESCs were enriched based on collagen type IV adhesiveness. They exhibited many characteristics predicted of ESCs such as quiescence, small blast-like morphology, and the greatest long-term cell regenerative output in vitro.β1-integrin, CK19 and PCNA expression were positive. Almost all cells from a single colony wereα6briCD71dim staining by immunofluorescence.2. The proliferation and migration of ESCs dependent on SDF-1αconcentration. The promoting effect of SDF-1αwas the most significant in 100ng/ml. AMD3100 was able to effectively block the effect of SDF-1α.3. Using ESCs as seed cells, we successfully constructed a full-thickness skin frostbite model in the three-dimensional skin model. HE staining showed the well developed skin structure in normal area, as well as thinner skin and increased ESCs in wound margin of frostbite. As shown by Immunohistochemical staining, the expression of SDF-1 in the wound margin increased with time and peaked in day 7, followed by a rapid decline. There was on SDF-1 expression in wound area in the late healing stage. Instead of single-sheet and scattered in the basal layer just like in normal skin, the distribution of ESCs was tend to gather around wound. Theβ1-integrin-positive cells appeared in both basal and upper layers of skin. The newborn epidermal tissue was thicker than normal tissue.Conclusion:1. ESCs can be selected and cultured in vitro successfully based on collagen type IV adhesiveness.2. Exogenous SDF-1αcould promote the proliferation and migration of ESCs by interacting with its receptor CXCR4.3. A controllable expression of SDF-1 was detected in wound margin during skin repair on TDSE. SDF-1 could induce the ectopic distribution of ESCs with its chemotaxis, and accelerate skin epithelization with the proliferation promoting and chemotactic effects.
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