| Co-Investigator(Kenkyū-buntansha) |
HIGASHIYAMA Shigeki OSAKA UNIVERSITY, BIOCHEMISTRY, ASSOCIATE PROFESSOR, 医学部, 助教授 (60202272)
HANAKAWA Yasushi EHIME UNIVERSITY, DERMATOLOGY, ASSISTANT PROFESSOR, 医学部, 助手 (90284398)
SHIRAKATA Yuji EHIME UNIVERSITY, DERMATOLOGY, ASSISTANT PROFESSOR, 医学部, 助手 (50226320)
HASHIMURA Etsuro OTSUKA PHARMCEUTICAL CO. RESEARCHER
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| Research Abstract |
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.
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