RESEARCH FOR ARTIFICIAL SKIN MASS PRODUCTION SYSTEM INTEGURATED WITH FOREIGN GENE

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Preparation of three dimensional reconstituted skin : Fibroblasts from human skin were cultured in collagen gel, then keratinocytes were seeded on the top of the gel. When the keratinocytes reached to confluent, the co-culture was incubated under air liquid interface. Stratified three dimensional reconstituted skin were analyzed by immunohistochemically or by electronmicroscopy. The cell adhesion molecules such as desmoglein1, desmoglein3, desmoplakin, E-cadherin, and beta 1 integrin, or involucrin as a marker of differentiation were fully expressed in the three dimensional reconstituted skin. Hemidesmosome was well formed, bullous pemphigoid antigen and beta 4 integrin were expressed, but anchoring fibrils or lamina densa were not so well developed. We could produce large size skin equivalent 75 mm in diameter.Preparation of adenovirus vector : We integrated c DNA of HB-EGF or GEP into replication-deficient adeno virus, produced large amounts of vector, and purified. Keratinocytes were infected with these adenovirus vectors and the expression was analyzed by immunohistochemistry. HB-EGF protein was detected in almost all keratinocytes. In the three dimensional reconstituted skin, we injected adenovirus vector between the epidermis and dermis. After 2 days, HB-EGF and GFP protein were expressed in the epidermis. Those expression persisted until 7 days, and noticed in all layer of the epidermis.