| Project/Area Number |
08407021
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Dermatology
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| Research Institution | EHIME UNIVERSITY |
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Principal Investigator |
HASHIMOTO Koji EHIME UNIVERSITY, DERMATOLOGY, PROFESSOR, 医学部, 教授 (00110784)
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| Co-Investigator(Kenkyū-buntansha) |
TOHYAMA Mikiko EHIME UNIVERSITY, DERMATOLOGY, ASSISTANT PROFESSOR, 医学部附属病院, 助手 (60263935)
SHIRAKATA Yuji EHIME UNIVERSITY, DERMATOLOGY, ASSISTANT PROFESSOR, 医学部, 助手 (50226320)
NAKAOKA Hiroki EHIME UNIVERSITY, DERMATOLOGY, ASSISTANT PROFESSOR, 医学部, 講師 (30172266)
TAMAI Katsuto HIROSAKI UNIVERSITY, DERMATOLOGY, ASSISTANT PROFESSOR, 医学部, 助教授 (20236730)
HANAKAWA Yasushi EHIME UNIVERSITY, DERMATOLOGY, ASSISTANT PROFESSOR, 医学部, 助手 (90284398)
宮内 俊次 愛媛大学, 医学部, 助教授 (10166118)
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| Project Period (FY) |
1996 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥8,200,000 (Direct Cost: ¥8,200,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1998: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1997: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Keywords | ADENOVIRUS VECTOR / EPIDERMAL KERATINOCYTE / BETA GALACTOSIDASE / GENE TRANSFER / SERUM FREE CULTURE / 293 CELL / MOI / STEM CELL / 無血清培養法 / 三次元培養皮層 / 三次元培養皮膚 |
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| Research Abstract |
To identify the stem cell, high beta 1 integurin was used as a marker, but the most important characteristics of stem cell is capacity of its proliferation. So, we tested how long keratinocytes are able to proliferate. Keratinocytes obtained from 15 healthy volunteers ranged between 1 to 70 years old continued to be cultured until they were reached to senescence. Population doubling time (PD) was ranged widely from 5.7 to 45.2. This indicated the number of stem cell was different by donor. PD tended to be higher in younger donor, but PD of one strain from 45 years old female was 41.3. These strains were divided into three groups such as short, intermediate and long, based on their PD. We suggest that it will be possible to analyze epidermal stem cells more precisely using this classification. 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 electron microscopy. The cell adhesion molecules and differentiation markers were fully expressed in the three dimensional reconstituted skin. Basement membrane components as hemidesmosome, bullous pemphigoid antigen and beta 4 integurin were formed and expressed sufficiently, but anchoring fibrils or lamina densa were poorly developed. Moreover, we transfer foreign genes into keratinocytes using replication-deficient adenovirus vector. Beta galactosidase was detected in almost all keratinocytes. In the three dimensional reconstituted skin, beta galactosidase was expressed in the epidermis after 5 days, but its expression was higher in the upper layer than the basal layer. The toxicity was not found up to MOI10.
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