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血中遊離ヒストンの肺移植後グラフトに与える影響の検討

中尾, 啓太 東京大学 DOI:10.15083/0002004558

2022.06.22

概要

【背景】
肺移植は他の治療が奏功しない進行性の呼吸器疾患に対して行われる医療であり、現在全世界で年間4000例以上、国内で60例以上行われている。しかしながら、その5年生存率は全世界で約半数、国内でも約7割である、術後1年以上では慢性拒絶反応が最多の原因であるが、術後30日以内では移植肺機能不全が多いことが知られている。その中で、術後72時間以内に起きるX線透過性低下を伴う肺水腫であるprimary graft dys function(PGD)に今研究では着目した。PGDは肺移植の再灌流後に生じるacute lung injury(ALI)の一種であるとされているが、未だに真の病態生理の解明や治療法の開発は行われておらず、同所性肺移植を伴う動物実験モデルも広く用いられているものはない。今研究では、脳卒中・外傷など組織傷害を伴う種々の病態で血中に遊離し、damage associated molecular patterns (DAMPs)の一種として働くhistoneを用いた。histoneは全身にdanger signalを伝える働きを持ち、血管内皮傷害・臓器障害作用や凝固促進作用を持つことが知られている。また、histoneの作用を中和する方法として、disseminated intravascular coagulation(DIC)の治療薬として臨床使用されており、抗histone作用を持つことが知られている遺伝子組み換え型ヒトトロンボモジュリンアルファ(rhTM)を用いた。

脳死肺移植ドナーが脳死に至る過程での、histone関連肺障害が肺移植後PGDの原因の一つとなっているという仮説を立て、ラット片肺移植モデルを用いて、PGD動物実験モデルの作成およびその治療の可能性を検討した。

【方法】
仔牛胸腺由来histone40mg/kg(Histone群)、仔牛胸腺由来histone40mg/kgおよびrhTM3mg/kg(Histone+rhTM群)、リン酸緩衝生理食塩水(PBS)(Control群)を経静脈投与したWistarラットをドナーとして用いた。各薬剤投与3時間後に心肺ブロックを摘出し、左肺をカフ法で別の個体の同等の体格のWistarラット(レシピエント)へ同所性肺移植を行い、再還流後4時間まで観察した。ドナー(実験1)およびレシピエント(実験2)の評価は、血液ガス分析、X線写真、酸素化と透過性でつけられるPGDgrade、肺組織のrealtimeRT-PCR(GAPDHに対するCXCL-1、TNFの相対発現)、病理組織学的検討(Hematoxylin-Eosin(HE)染色、Phospho Tungstic Acid-Hematoxylin(PTAH)染色、von Willebrand factor(vWF)免疫染色)、血漿タンパク質分析(Enzyme-Linked Immuno Sorbent Assay法、マルチプレックス法)を用いて行った。統計解析は全てGraphPadPrism7(Graphpad Software. San Diego, CA, USA)を使用した。得られたデータは、平均±標準偏差で表記し、2群間の検定は、Mann-Whitney検定、3群間の検定は、Kruskal-Wallis検定で行った。対応のある3群間の比較は、Friedman検定を用いた。P値<0.05を統計学的に有意と解釈した。

【結果】
実験1(ドナーの評価)ラットへのhistone投与によって酸素化・X線の透過性の変化は見られなかった。しかしながら、血漿タンパク質・肺組織RNA相対発現では血管内皮傷害を起こしていることが観察された。また、病理所見では、血栓形成は認めなかったが、浮腫性変化を認めた。ラットへのhistoneとrhTMの投与によって酸素化・X線の透過性の変化は見られなかった。血漿タンパク質(syndecan-1、PAI-1など)・肺組織RNA相対発現(CXCL-1)では有意差はないものの、histone単独投与と比較して、histoneとrhTMの投与は血管内皮傷害が軽減している傾向にあった。また、病理所見でもhistoneとrhTMの投与を受けている群は、浮腫性変化を認めなかった。

実験2(レシピエントの評価)レシピエント体内に同所性肺移植を行ったグラフトへの影響の検討では、酸素化はControl群・Histone+rhTM群では再灌流後に経時的に回復が見られたが、Histone群では経時的回復は見られず、低値となった。また、PGDgradeに関しても、Control群・Histone+rhTM群と比較して、Histone群では有意にgradeが高かった。病理所見などからは、炎症細胞浸潤による鬱血がその病態であったことが示唆されるが、病理所見のlung lesion scoreに有意差はなかった。

【結語】
今研究では、ドナーに経静脈投与したhistoneによる肺傷害は、我々が臨床で肺移植のグラフトを評価する際に用いている方法では評価できないが、RNA相対発現などでは変化を認めた。レシピエントに同所性片肺移植を行うことによりグラフトに酸素化の低下およびX線透過性の低下を惹起することが証明され、PGD動物実験モデルの作成を行うことができた。また、その変化はrhTMの投与により軽減され、ドナー体内での治療によりグラフトPGDの軽減ができる可能性が示唆された。

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