| 研究課題/領域番号 |
14F04106
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| 研究機関 | 岡山大学 |
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研究代表者 |
松本 卓也 岡山大学, 医歯(薬)学総合研究科, 教授 (40324793)
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| 研究分担者 |
HARA Emilio 岡山大学, 医歯(薬)学総合研究科, 外国人特別研究員
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| 研究期間 (年度) |
2014-04-25 – 2016-03-31
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| キーワード | 軟骨 / 合成微小環境 / DNAメチル化 / 石灰化 |
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| 研究実績の概要 |
In this study, we are attempting to synthesize a biomimetic microenvironment for fabrication of cartilage in vitro, using materials (e.g., hydrogels) and biological tools, including overexpression of proteins in cells. We hypothesized that chondrogenic differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (hBMSCs) could be regulated by DNA methyltransferases (DNMTs) on specific genes involved in the promotion or suppression of chondrogenesis.
To study the effect of DNMTs on chondrogenesis of hBMSCs, we plan to transfect DNMTs overexpression vectors in the cells, and culture the cells in a 3D micromass culture system.
Since in vitro synthesized cartilage tissue generally undergoes through mineralization, we are also attempting to understand the mechanisms involved in the mineralization process during initial bone formation.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Initially, we analyzed the expression pattern of DNMTS during normal process of chondrogenesis in vitro, and found that DNMT3A and DNMT3B were significantly upregulated. Then, we transfected DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) overexpression vectors in hBMSCs, and induced the cells to differentiate into chondrocytes by culturing the cells in a 3D micromass culture system in chondrogenesis-inducing medium for 21 days. The results showed that overexpression of DNMT3A induced a strong increase in the deposition of cartilage matrix, as detected by toluidine blue staining of histological sections of the micromasses. Similar to DNMT3A, overexpression of DNMT3B also increased the expression of cartilage markers, aggrecan and collagen type II, however, to a less extent.
To analyze the process of mineralization from chondrocytes, we observed the formation of minerals during secondary ossification of mouse femur. We found that mineralization occurs in the initial 7 days after birth, and it progresses relatively quickly until the second week after birth.
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| 今後の研究の推進方策 |
We plan to identify the mechanism of how DNMT3A and DNMT3B regulates chondrogenesis of hBMSCs; more specifically, to identify the sites methylated by DNMT3A and DNMT3B in the promoter or enhancer region of key genes, including SOX9, ACAN, COL2A1.
We also plan to identify the types of mineral formed in the initial process of bone formation, and analyze the maturation process of mineral crystals.
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