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大学・研究所にある論文を検索できる 「Ⅲ度熱傷マウスの全層皮膚切除部位に対する、ハイパードライヒト乾燥羊膜とヒト羊膜間葉系幹細胞を用いた研究」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

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Ⅲ度熱傷マウスの全層皮膚切除部位に対する、ハイパードライヒト乾燥羊膜とヒト羊膜間葉系幹細胞を用いた研究

小橋 大輔 富山大学

2021.03.23

概要

〔目的〕
重症熱傷患者における死因の50-60%は敗血症によるものであり、その治療戦略として感染制御は最重要といえる。重症熱傷患者では、全層皮膚切除部位の正常肉芽増生を促し、創部感染制御に有効に働く創傷被覆材の使用が早期植皮・閉創につながり、救命率向上の一助となる可能性が高い。しかしながら現在のところ、植皮術までの全層皮膚切除部位に対する被覆材として確立されたものはない。羊膜は古くから被覆材として利用されており、本邦では既に眼科領域で羊膜移植術として利用されている。富山大学では生羊膜を特殊な方法で乾燥・滅菌し、ハイパードライヒト乾燥羊膜(HD-AM)として臨床応用する方法を確立した。また、羊膜から間葉系細胞を分離、培養する方法を確立し、幹細胞の性質を有する細胞株(human amniotic mesenchymal cells;HAMα)を樹立した。本研究では、III度熱傷マウスモデルの全層皮膚切除部位に、HD-AMを被覆材としてHAMαを添加することで、早期の肉芽形成が促進されるかについて組織学的、免疫組織学的、分子生物学的に検証した。

〔方法並びに成績〕
ドナーより採取した生羊膜を遠赤外線連続照射、減圧、マイクロ波間欠照射により乾燥させ、γ線照射により滅菌を行うことでHD-AMを作製し、また、37℃、5%CO2の条件下で培養したHAMαを、6x106 cells/mLの濃度になるようにPBS溶液で希釈(HAMα-PBS溶液)した。背部の除毛を行ったマウスを 90℃の熱湯を10秒間暴露させ、同部位の全層皮膚切除を行い開放創とした。開放創にPBS溶液を滴下した群(control群)、PBS滴下に加え、HD-AMを置いた群(HD-AM群)、HAMα-PBS溶液を滴下した群(cell群)、HAMα-PBS溶液の滴下に加え、HD-AMを置いた群(HD-AM/cell群)の4群に分けて術後1日(POD1)、4日(POD4)、7日(POD7)に評価を行った。遺伝子の発現については、ハウスキーピング遺伝子の安定性を確認するため、GAPDH、RPLP1、RPS18について測定を行い、 Ct(Threshold Cycle)値を算出した。また、Ct値の誤差がHAMαに起因するかについて、human β2-M(β2-microglobulin)の発現量を用いて検討した。さらに、inducible nitric oxide synthase (iNOS)、CD163、IL-6、IFN-γ、IL-10、COX-2について、qRT-PCRを行い、発現量の測定を行った。HAMαは移植前にCell Tracker™ を用いて標識し、移植細胞の局在を確認した。Azan染色にて組織染色を行い、 CD31、α-SMA、IL-6、IL-10、CD163については免疫組織染色を行いその局在について評価した。
POD1、POD4においてはHAMαが残存していたことが確認され、POD4ではすべての群で肉芽組織の形成を認めたが、cell群およびHD-AM/cell群における肉芽組織の形成が顕著であった。POD7ではHD-AM群、cell群、HD-AM/cell群において、肉芽組織内には毛細血管だけでなく、平滑筋を備えた毛細血管以上の血管も形成された。また、 HD-AM/cell群においては、血管形成が他の群よりも減弱していることが明らかとなった。このことから、早期の血管形成に HD-AMとHAMαの双方が関与すると同時に、過剰な血管形成を抑える働きもあることが示唆された。ハウスキーピング遺伝子についてはCt値の差が最も小さいRPS18を用いて試料の標準化を行った。また、cell群、HD-AM/cell群においてhumanβ2-Mの発現を認めたもののその発現量はわずかであり、 HAMαによってCt値の誤差が生じている可能性は低いと考えられた。
HD-AM群、cell群、HD-AM/cell群においてiNOSや、IFN-γ、COX-2、IL-6といった炎症性サイトカインが早期に上昇し、POD7ではこれらの発現が低下する傾向を示した。 POD7では組織修復に関わるtype2マクロファージの発現を表す CD163、抗炎症サイトカインであるIL-10の発現が増加していることから、HD-AM群、cell群、HD-AM/cell群では増殖期から組織再構築期に移行し、コラーゲンの産生が高まったと考えられる。一方、control群ではPOD7において炎症性サイトカイン、抗炎症性サイトカインの発現は低いものの、組織染色において血管形成および肉芽形成がほとんど認められないことから、適切な創傷治癒が起こっている可能性は低いと考えられた。
以上より、創傷治癒の促進には HD-AMの細胞誘導機能、足場としての役割に加えて HAMαが分泌するサイトカインが必要と考えられた。

〔総括〕
重症熱傷における創傷被覆材としてのHD-AMならびにHAMαの有効性について検討した。HD-AMにHAMαを加えたことで、 HD-AM群よりも早く、かつ厚い肉芽を形成することが可能となった。肉芽は形態的に線維芽細胞、血管内皮、マクロファージなどで構成された良好な肉芽であり、HD-AMを単独で使用するよりも HAMαを添加することで早期の血管増殖が可能となった。HD-AMにHAMαを組み合わせた被覆材は III度熱傷後の全層皮膚切除部位に対する早期の肉芽増生に有用であり、重症熱傷患者の救命率向上に向けた治療技術になり得る。
重症熱傷における創傷被覆材としてのHD-AMならびにHAMαの有効性について検討した。HD-AMにHAMαを加えたことで、 HD-AM群よりも早く、かつ厚い肉芽を形成することが可能となった。肉芽は形態的に線維芽細胞、血管内皮、マクロファージなどで構成された良好な肉芽であり、HD-AMを単独で使用するよりも HAMαを添加することで早期の血管増殖が可能となった。HD-AMにHAMαを組み合わせた被覆材は III度熱傷後の全層皮膚切除部位に対する早期の肉芽増生に有用であり、重症熱傷患者の救命率向上に向けた治療技術になり得る。

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