| Project/Area Number |
20591347
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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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 |
KAMEDA Kenji Ehime University, 総合科学研究支援センター, 講師 (60363264)
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|---|
| Co-Investigator(Kenkyū-buntansha) |
SHIRAKATA Yuji 愛媛大学, 大学院・医学系研究科, 講師 (50226320)
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| Project Period (FY) |
2008 – 2010
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| Project Status |
Completed (Fiscal Year 2010)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2010: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2009: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2008: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Keywords | 皮膚炎症 / 再生学 / 表皮幹細胞 / 羊膜 / SP細胞 / セルソーター / 三次元培養 |
|---|
| Research Abstract |
In order to develop an easy method for constructing a three-dimensional cultured skin, we focused on epidermal stem cells and amnion membrane. We analyzed the condition for side population cells as stem cells, and clarified that ten microgram per ml of Hoechst dye, 60 min treatment at 37 degree are best for side population staining. Then we examined side population cell rate among different passaged cells, and demonstrated that side population cell ratio decreased along with passaging. Next we analyzed about amnion membrane. Amnion membrane was overlaid on type I collagen gel embedded with fibroblasts; then, normal human keratinocytes were seeded on the epithelial side of the amnion membrane. When the keratinocytes reached confluence, the three-dimensional cultured skin was lifted to the air-liquid interface and cultured for up to 3 weeks. Samples were harvested at different times and manipulated for morphological, immunohistochemical, and ultrastructural investigation. In the three-dimensional cultured skin with amnion, the epidermis was better stratified, with more compact, polarized, columnar basal cells, and the expression of differentiation and proliferation markers was more similar to that of normal human skin than was that of the three-dimensional cultured skin without amnion membrane. A more continuous basement membrane and better-developed hemidesmosomes were found in the three-dimensional cultured skin with amnion. It is possible to construct a better cultured skin using amnion membrane.
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