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部分切除肺から得た自家細胞をもとに脱細胞化技術で作成した再生肺の移植の検討

柳谷, 昌弘 東京大学 DOI:10.15083/0002005135

2022.06.22

概要

死因の主要な地位を占める末期肺疾患の有望な治療は同種他家肺移植であるが，拒絶などの問題がある．これらの問題解決のために，レシピエントと遺伝的に同一な細胞に基づいた肺再生が望まれる．臓器にある細胞を完全に除去し，細胞外マトリックスのみの構造物を構築する脱細胞化技術を用いて，我々は肺の臓器再生を試みた．具体的にはドナーから得た臓器を脱細胞化して得られたスキャフォールドに，レシピエント由来の自家細胞を再配置することで臓器再生を行うというものである．

我々は，大動物であるブタモデルにおいて，脱細胞化技術を用いてレシピエント由来の自家細胞をもとに再生肺を構築し，左片肺移植を行ってInvivo機能の評価を行った．この検討の中で我々は次の4つの検討を行った．1つ目は脱細胞化に関する検討である．ドナーブタから得た肺グラフトにドデシル硫酸ナトリウムならびにTritonX-100を肺動脈に灌流する方法を用いて実際に脱細胞化が実現されたかについて検証した．さらに灌流前に気管支内に生理食塩水を注入することでより効果的な脱細胞化が得られるかについても検証した．2つ目の検討はレシピエントブタに対して肺部分切除を行い，自家細胞の分離培養を行うというものである．我々はKRT5陽性p63陽性気道上皮細胞ならびに血管内皮細胞の分離培養を試み，Characterizationを行った．3つ目の検討は，脱細胞化肺スキャフォールドに自家細胞を培養し，実際に自家細胞による再細胞化がなされたかについて検証した．最後に，再細胞化して得られた再生肺をレシピエントブタに左片肺移植を行い4時間生存させ，ガス交換能を調べた．再生肺の左片肺移植のコントロール群として同種他家肺移植，ならびに脱細胞化肺スキャフォールドの左片肺移植を設定した．

1つ目の脱細胞化の結果について，脱細胞化後の肺組織のヘマトキシリンエオジン染色では細胞成分をほとんど認めなかった．さらに正常肺グラフトに比して脱細胞化肺スキャフォールドはDNA量が有意に低かった（35.1±17.1vs.5300±2530μg/mgwet tissue,P<0.001）．さらにこの検討の中で我々は，ドデシル硫酸ナトリウムならびにTritonX-100の灌流前に気管支内に生理食塩水を注入することで，より効果的な脱細胞化が実現できることを示した．気管支内生理食塩水を注入した群では，そうでない群に比して有意にスキャフォールドのDNA量が有意に低かった（21.6±5.46vs.48.7±12.3μg/mgwet tissue,P<0.05）．

2つ目の検討である自家細胞の分離培養であるが，我々は144mg-1376mgの肺を得て，3週間の培養を経て，気道上皮細胞は平均19.4×105個，血管内皮細胞は平均34.9×105個まで得ることができた．気道上皮細胞は免疫組織化学染色ならびにフローサイトメトリーでほぼ全ての細胞においてKRT5ならびにp63陽性細胞であることを示した．さらに血管内皮細胞も免疫組織化学染色ならびにフローサイトメトリーでほぼ全ての細胞においてCD31陽性細胞であることを示した．

つ目の検討である脱細胞化肺スキャフォールドに対する自家細胞の培養について，我々は血管内皮細胞を2日間，気道上皮細胞を1日間かけて培養した．再細胞化した肺組織のヘマトキシリンエオジン染色で散在性に細胞を確認した．さらに免疫組織化学染色でKRT5陽性p63陽性気道上皮細胞ならびにCD31陽性血管内皮細胞の存在を確認した．4つ目の検討として，再細胞化して得られた再生肺を左片肺移植した．再生肺移植を行った3例ならびに同種他家肺移植を行った3例は全例4時間以上生存したものの，脱細胞化肺スキャフォールドを移植した2例は移植後30分以内に死亡した．移植された再生肺グラフトは再換気直後から著明な嚢胞性変化を示した．再生肺移植群は同種他家肺移植群と比較して移植後4時間酸素ガス交換能はほぼ同等であった．しかしながら，再生肺移植群の二酸化炭素ガス交換能は，同種他家肺移植に比して悪く，移植後4時間の時点で二酸化炭素ガス交換能は低い傾向にあった（3.93±3.61mmHgvs.21.1±13.2mmHg,P=0.096）．

脱細胞化肺スキャフォールドを移植した2例の肺組織を検証すると，肺出血像を呈していた．さらに移植後の再生肺グラフトの胸膜損傷部を免疫組織化学染色で調べるとエラスチンが保持されていなかったことが明らかとなった．

本研究は，部分切除から得たレシピエント由来の自家細胞をもとに，脱細胞化技術を用いて構築した再生肺の左片肺移植は実現可能であることを示した．脱細胞化スキャフォールドを左片肺移植した場合には肺出血で死亡に至ったことを考えると，レシピエント由来の自家細胞で血管を再細胞化することでレシピエント由来の血流に耐えうる再生肺が構築できたと考えることができる．しかしながら再生肺は嚢胞性変化や二酸化炭素ガス交換能不良といった結果をきたし，臨床応用にはほど遠いことを示唆した．これらの変化は，脱細胞化過程におけるエラスチン喪失が一因と考えられた．

今後の肺再生研究を行う上で次の2点が重要と考えられた．1つはエラスチンをはじめとする細胞外マトリックスタンパクの保持ができるような脱細胞化の実現である．我々の用いたドデシル硫酸ナトリウムならびにTritonX-100以外の手法も模索する必要がある．2つ目は細胞についての検討である．今回我々が使用した気道上皮細胞ならびに血管内皮細胞だけで本当に肺再生が実現できるのか，細胞の質ならびに量の面から今後検討すべき余地は大いにある．

本研究は肺再生研究の課題を提示し，大きな礎になると考える．

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