| The skin is considered as the largest organ of human body. The delicate and complicated structure and multiple function of the skin make it play an important role in secrete, sense, metabolism, protection and so on. If the skin is injured or defects, the stability of internal environment of the body will be impacted and severe complications, such as infection, disorder of fluid and electrolytes, as well as immunologic impairment, will occur. The common repair methods of skin defect are autografting, allografting and xenografting. The clinical experience has shown that the formation of hyperplastic scar after skin grafting is increasing as the dermal thickness of skin grafts is decreasing. For this reason, there must be enough dermis in skin grafts to repair the skin defect; the best is full thickness skin graft or skin flap transplantation. But it is impossible for the patients with massive skin defects or burn injuries to cover all the wounds with full thickness skin grafts or flaps because conventional methods are limited by the availability of donor sites. To study an ideal skin substitute has become one of major problems in the clinical treatment. With the emergence and development of tissue engineering, the establishment of tissue-engineering skin will open up broad prospects for repair of severe skin injuries.Since Rheinwald and Green established the method of keratinocyte culture successfully in 1975, the technique of skin culture has been improved greatly. Scientists have not only used cultured keratinocyte sheets in the treatment of massive burns or chronic ulcer, but also combined cultured keratinocytes with dermal substitute to construct composite skin successfully. So far there have been several commercial and industrial tissue-engineering skins to be used. Although there has been tremendous step forward in this area, many problems still need to be solved, such as relative difficulty to get cadaver skin, antigenicity of dermis, culture stability of keratinocytes, vescularization and growth regulation of composite skin. Therefore, it is necessary to study further on tissue-engineering skin thoroughly. [Objective]According to comparability in structure and metabolism between pigskin and mankind skin, using Trypsin digestion and repeated freeze-thaw cycles to prepare acellular xenodermis as a cell scaffolding. To construct eukaryotic expression plasmid ofhuman platelet-derived growth factor-B, then transfected to flbroblasts. To study further on tissue-engineering skin by establishment of the composite skin with combining cultured keratinocytes, acellular pig dermis and flbroblasts which have been transfected with PDGF gene.[Methods]1. The Partial-thickness porcine skins were enzymolysed with 0.05 percent Trypsin at 4 for a night, and then the epidermis was stripped off mechanically under sterile conditions. The remaining dermis was thoroughly washed in PBS to remove the cell and the cell debris. After that the dermis was treated again with trypsin at same concentration for one hour at 37 , rinsed with PBS. The obtained dermis was placed into refrigerator at 4 and -74 respectively for 2h, followed by thawing at room temperature. This procedure was repeated for three times, and then well washed in sterile PBS with continuous shaking to eliminate remnants of cells. The prepared acellular dermis was examined macroscopically and microscopically with HE staining and immunohistochemical staining, bacteria culture test and cytotoxicity test. It was also examined by implanted on the fascia beneath the skin flap of the rat.2. Full thickness skins were cultivated in the condition of asepsis from approximately 1-day-old postnatal SD rats, soaked in fresh D-hanks' solution with 0.01% trypsin at 4 for 18-24 hours and separated into epidermal layer and dermal layer with low temperature enzyme digestion. The basal lamina cells between the two layers were scraped off and pure keratinocytes were obtained by using gradient density centrifugation (DGC). Keratinocyt...
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