羊膜の基質上で培養したヒト骨膜由来細胞シートの開発
目的 : 羊膜は抗炎症・感染抑制作用などを有し, 種々の手術療法に用いられ, 移植材料としてだけでなく培養基質としても高い有用性・有効性が注目されている. われわれはこの羊膜に注目し, 羊膜を基質とした各種培養細胞シートを作製し, 一部では臨床応用を実施しており新たな再生医療的治療法としての有効性を示してきた. 近年, 骨膜由来の細胞をシート状に培養して移植し, 歯周組織再生を試みる研究が散見される. 今回, 新規骨膜由来細胞シートの作製を念頭に, 羊膜上にて骨膜由来細胞の培養を行い, 同培養シートのin vivoにおける細胞動態について免疫組織学的な検討を加えた. 材料と方法 : 羊膜は帝王...
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| Published in | The Japanese Journal of Conservative Dentistry Vol. 59; no. 5; pp. 394 - 401 |
|---|---|
| Main Authors | , , , , , , , , |
| Format | Journal Article |
| Language | Japanese |
| Published |
特定非営利活動法人 日本歯科保存学会
2016
The Japanese Society of Conservative Dentistry |
| Subjects | |
| Online Access | Get full text |
| ISSN | 0387-2343 2188-0808 |
| DOI | 10.11471/shikahozon.59.394 |
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| Abstract | 目的 : 羊膜は抗炎症・感染抑制作用などを有し, 種々の手術療法に用いられ, 移植材料としてだけでなく培養基質としても高い有用性・有効性が注目されている. われわれはこの羊膜に注目し, 羊膜を基質とした各種培養細胞シートを作製し, 一部では臨床応用を実施しており新たな再生医療的治療法としての有効性を示してきた. 近年, 骨膜由来の細胞をシート状に培養して移植し, 歯周組織再生を試みる研究が散見される. 今回, 新規骨膜由来細胞シートの作製を念頭に, 羊膜上にて骨膜由来細胞の培養を行い, 同培養シートのin vivoにおける細胞動態について免疫組織学的な検討を加えた. 材料と方法 : 羊膜は帝王切開時の胎盤より採取したものを研究に供し, 骨膜組織は口腔外科手術時の粘膜骨膜弁作製時に, 歯槽骨上にある組織を骨膜組織として採取した. 得られた骨膜組織の初代培養を行い, 3～4代継代したものを骨膜由来細胞とした. 得られた細胞を, 上皮細胞を剝離・除去した羊膜上に播種し3週間の培養を行った. 上記にて得られた培養シートをヌードマウス腎被膜下へ移植し, 4週間後に摘出, H-E染色, ならびに免疫染色を行った. 成績 : 骨膜由来細胞は羊膜上にて層状構造を示し, 免疫染色像では細胞増殖マーカーであるKi-67, 間葉系細胞マーカーであるvimentin, 骨芽細胞マーカーであるosteocalcinの発現が認められた. また, デスモソームのマーカーであるdesmoplakinおよびタイト結合のマーカーであるZO-1が発現し, 細胞シートを形成していた. また, 移植後もvimentin, osteocalcinの発現を認め, その性質を保持していた. 結論 : 骨膜由来細胞は羊膜上で1枚の細胞シートを形成して増殖しており, 羊膜は培養に適当な基質であることが示唆された. 骨膜には骨芽細胞や骨細胞へ分化する能力をもつ細胞が含まれているとされ, 細胞シートにて骨芽細胞が産生するタンパクの発現を認め, in vivoの環境下でもその性質を保持していた. 今後, 長期間移植後の検討が必要と思われるが, 羊膜を用いた骨膜由来細胞シート作製が可能であることが示された. |
|---|---|
| AbstractList | Purpose: An amniotic membrane (AM) with anti-inflammatory and anti-infective effects is attracting attention for its usefulness and effectiveness as a culture substrate and graft material, and is currently used in a number of surgical procedures. We previously prepared various cell sheets cultured with AM as the substrate, and demonstrated that some sheets may be effectively used in clinical settings for regenerative medicine. In recent studies, cultured cell sheets derived from the periosteum were transplanted in order to regenerate periodontal tissues. In the present study, periosteal-derived cells (PDCs) were cultured on AM to generate novel periosteal cell sheets, and the kinetics of the cell sheets were immunohistochemically investigated in vivo. Methods: AM was removed from the placenta during cesarean section, and periosteal tissues on alveolar bones were collected upon the creation of a mucoperiosteal flap during oral surgery. Periosteal tissues were primarily cultured, followed by three to four passages to obtain PDCs. These cells were seeded on AM, and the epithelial cells that developed were scraped off and then cultured for three weeks. The resulting culture sheets were transplanted under the renal capsules of nude mice, and were removed after 4 weeks for H-E staining and immunostaining. Results: PDCs showed a layered structure on AM. Immunostaining images revealed the expression of Ki-67 (a cell proliferation marker), vimentin (a mesenchymal cell marker), and osteocalcin (an osteoblast marker). Desmoplakin (a desmosome marker) and ZO-1 (a tight junction marker) were also expressed with cell sheet formation. Vimentin and osteocalcin were expressed even after transplantation, and the properties of the cell sheets were maintained. Conclusion: PDCs proliferated on AM, forming single cell sheets, which demonstrates that AM is suitable as a culture substrate. The periosteum contains cells that have the ability to differentiate into osteoblasts and osteocytes. The cell sheets expressed osteoblast proteins, which were maintained even in an in vivo environment. Thus, PDC sheets may be prepared with AM. A long-term transplantation study will be conducted in the future.
目的 : 羊膜は抗炎症・感染抑制作用などを有し, 種々の手術療法に用いられ, 移植材料としてだけでなく培養基質としても高い有用性・有効性が注目されている. われわれはこの羊膜に注目し, 羊膜を基質とした各種培養細胞シートを作製し, 一部では臨床応用を実施しており新たな再生医療的治療法としての有効性を示してきた. 近年, 骨膜由来の細胞をシート状に培養して移植し, 歯周組織再生を試みる研究が散見される. 今回, 新規骨膜由来細胞シートの作製を念頭に, 羊膜上にて骨膜由来細胞の培養を行い, 同培養シートのin vivoにおける細胞動態について免疫組織学的な検討を加えた. 材料と方法 : 羊膜は帝王切開時の胎盤より採取したものを研究に供し, 骨膜組織は口腔外科手術時の粘膜骨膜弁作製時に, 歯槽骨上にある組織を骨膜組織として採取した. 得られた骨膜組織の初代培養を行い, 3～4代継代したものを骨膜由来細胞とした. 得られた細胞を, 上皮細胞を剝離・除去した羊膜上に播種し3週間の培養を行った. 上記にて得られた培養シートをヌードマウス腎被膜下へ移植し, 4週間後に摘出, H-E染色, ならびに免疫染色を行った. 成績 : 骨膜由来細胞は羊膜上にて層状構造を示し, 免疫染色像では細胞増殖マーカーであるKi-67, 間葉系細胞マーカーであるvimentin, 骨芽細胞マーカーであるosteocalcinの発現が認められた. また, デスモソームのマーカーであるdesmoplakinおよびタイト結合のマーカーであるZO-1が発現し, 細胞シートを形成していた. また, 移植後もvimentin, osteocalcinの発現を認め, その性質を保持していた. 結論 : 骨膜由来細胞は羊膜上で1枚の細胞シートを形成して増殖しており, 羊膜は培養に適当な基質であることが示唆された. 骨膜には骨芽細胞や骨細胞へ分化する能力をもつ細胞が含まれているとされ, 細胞シートにて骨芽細胞が産生するタンパクの発現を認め, in vivoの環境下でもその性質を保持していた. 今後, 長期間移植後の検討が必要と思われるが, 羊膜を用いた骨膜由来細胞シート作製が可能であることが示された. 目的 : 羊膜は抗炎症・感染抑制作用などを有し, 種々の手術療法に用いられ, 移植材料としてだけでなく培養基質としても高い有用性・有効性が注目されている. われわれはこの羊膜に注目し, 羊膜を基質とした各種培養細胞シートを作製し, 一部では臨床応用を実施しており新たな再生医療的治療法としての有効性を示してきた. 近年, 骨膜由来の細胞をシート状に培養して移植し, 歯周組織再生を試みる研究が散見される. 今回, 新規骨膜由来細胞シートの作製を念頭に, 羊膜上にて骨膜由来細胞の培養を行い, 同培養シートのin vivoにおける細胞動態について免疫組織学的な検討を加えた. 材料と方法 : 羊膜は帝王切開時の胎盤より採取したものを研究に供し, 骨膜組織は口腔外科手術時の粘膜骨膜弁作製時に, 歯槽骨上にある組織を骨膜組織として採取した. 得られた骨膜組織の初代培養を行い, 3～4代継代したものを骨膜由来細胞とした. 得られた細胞を, 上皮細胞を剝離・除去した羊膜上に播種し3週間の培養を行った. 上記にて得られた培養シートをヌードマウス腎被膜下へ移植し, 4週間後に摘出, H-E染色, ならびに免疫染色を行った. 成績 : 骨膜由来細胞は羊膜上にて層状構造を示し, 免疫染色像では細胞増殖マーカーであるKi-67, 間葉系細胞マーカーであるvimentin, 骨芽細胞マーカーであるosteocalcinの発現が認められた. また, デスモソームのマーカーであるdesmoplakinおよびタイト結合のマーカーであるZO-1が発現し, 細胞シートを形成していた. また, 移植後もvimentin, osteocalcinの発現を認め, その性質を保持していた. 結論 : 骨膜由来細胞は羊膜上で1枚の細胞シートを形成して増殖しており, 羊膜は培養に適当な基質であることが示唆された. 骨膜には骨芽細胞や骨細胞へ分化する能力をもつ細胞が含まれているとされ, 細胞シートにて骨芽細胞が産生するタンパクの発現を認め, in vivoの環境下でもその性質を保持していた. 今後, 長期間移植後の検討が必要と思われるが, 羊膜を用いた骨膜由来細胞シート作製が可能であることが示された. |
| Author | 大迫, 文重 足立, 圭司 遠藤, 悠美 浅井, 拓 足立, 哲也 西垣, 勝 雨宮, 傑 山本, 俊郎 金村, 成智 |
| Author_FL | Toshiro YAMAMOTO Tetsuya ADACHI Yumi ENDO Masaru NISHIGAKI Narisato KANAMURA Taku ASAI Takeshi AMEMIYA Fumishige OSEKO Keiji ADACHI |
| Author_FL_xml | – sequence: 1 fullname: Takeshi AMEMIYA – sequence: 2 fullname: Yumi ENDO – sequence: 3 fullname: Taku ASAI – sequence: 4 fullname: Tetsuya ADACHI – sequence: 5 fullname: Keiji ADACHI – sequence: 6 fullname: Masaru NISHIGAKI – sequence: 7 fullname: Fumishige OSEKO – sequence: 8 fullname: Toshiro YAMAMOTO – sequence: 9 fullname: Narisato KANAMURA |
| Author_xml | – sequence: 1 fullname: 浅井, 拓 organization: 京都府立医科大学大学院医学研究科 歯科口腔科学 – sequence: 1 fullname: 雨宮, 傑 organization: 京都府立医科大学大学院医学研究科 歯科口腔科学 – sequence: 1 fullname: 大迫, 文重 organization: 京都府立医科大学大学院医学研究科 歯科口腔科学 – sequence: 1 fullname: 金村, 成智 organization: 京都府立医科大学大学院医学研究科 歯科口腔科学 – sequence: 1 fullname: 足立, 哲也 organization: 京都府立医科大学大学院医学研究科 歯科口腔科学 – sequence: 1 fullname: 足立, 圭司 organization: 京都府立医科大学大学院医学研究科 歯科口腔科学 – sequence: 1 fullname: 山本, 俊郎 organization: 京都府立医科大学大学院医学研究科 歯科口腔科学 – sequence: 1 fullname: 遠藤, 悠美 organization: 京都府立医科大学大学院医学研究科 歯科口腔科学 – sequence: 1 fullname: 西垣, 勝 organization: 京都府立医科大学大学院医学研究科 歯科口腔科学 |
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| Copyright | 2016 特定非営利活動法人日本歯科保存学会 |
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| Publisher | 特定非営利活動法人 日本歯科保存学会 The Japanese Society of Conservative Dentistry |
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| Snippet | 目的 : 羊膜は抗炎症・感染抑制作用などを有し, 種々の手術療法に用いられ, 移植材料としてだけでなく培養基質としても高い有用性・有効性が注目されている.... Purpose: An amniotic membrane (AM) with anti-inflammatory and anti-infective effects is attracting attention for its usefulness and effectiveness as a culture... |
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| StartPage | 394 |
| SubjectTerms | amniotic membrane immunohistochemistry periosteal-derived cells 免疫組織化学 羊膜 骨膜由来細胞 |
| Title | 羊膜の基質上で培養したヒト骨膜由来細胞シートの開発 |
| URI | https://www.jstage.jst.go.jp/article/shikahozon/59/5/59_394/_article/-char/ja https://cir.nii.ac.jp/crid/1390001205522394112 |
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