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胚環境操作_2型糖尿病モデルMEMマウスにおける病態解明および大麦投与による病態抑制効果の検討

石山 詩織 山梨大学 DOI:info:doi/10.34429/00005149

2022.03.18

概要

胎生期低栄養環境は出生後の成熟期において、2 型糖尿病や心疾患を中心とした生活習慣病を発症するリスクを高めることが疫学研究で報告されており、胎生期栄養環境と産仔の成熟期における疾患のメカニズムについて、妊娠期食餌制限や子宮体動脈結紮などの動物モデルを用いて調べられてきた。しかし、胚環境操作による 2 型糖尿病モデルは作製されておらず、さらに糖尿病合併症の発症や抑制しうる食品因子についてはほとんど明らかにされていなかった。本論文では、胚発生初期である 2 細胞期胚を αMEM培地において体外培養し、産まれた産仔 MEM マウスが 2 型糖尿病の特徴である食後高血糖(第 1 章)、非アルコール性脂肪肝炎(第 2 章)や腎症(第 3 章)を呈するか、上記の病態は出生後の大麦投与により抑制されうるかを検討した。

第 1 章では、MEM マウスが日本人 2 型糖尿病患者で観察されるような食後高血糖を呈するか、また食後の末梢血白血球における炎症関連遺伝子の mRNA 発現が増大するかを検討した。離乳後から高脂肪食を摂取した MEM マウスは約 30 週齢時点において、通常培地 KSOM 培地にて体外培養され産まれ、離乳後から高脂肪食を摂取した対照マウスと比較して、軽度肥満にて 2 型糖尿病を発症することが明らかとなった。対照マウスと比較して、糖負荷後 15 分時において MEM マウスの血糖値が上昇し、糖負荷後 30分時および 120 分時にて上昇傾向が観察され、MEM マウスは食後高血糖を呈することが明らかとなった。さらに、糖負荷後 120 分時におけるMEM マウスの末梢血白血球における炎症関連遺伝子の mRNA 発現を測定した結果、Tnfa や Cd11b、Cd18 などの炎症性サイトカインおよび内皮接着因子インテグリンの mRNA 発現が、対照マウスと比較して増大した。すなわち、MEM マウスは、日本人型 2 型糖尿病と同様に軽度肥満にて食後高血糖を呈し、末梢血白血球での炎症関連遺伝子の mRNA 発現が高まるといった特徴を有するモデルマウスであることが示唆された。

第 2 章では、MEM マウスが糖尿病発症時に併発する合併症の一つである非アルコール性脂肪肝炎を呈するか、また出生後の大麦投与によりこの病態が抑制されうるかを検討した。その結果、MEM マウス肝臓では血管周囲において比較的大きな脂肪滴の蓄積およびコラーゲン線維の蓄積である顕著な線維化病態が観察され、定量化した脂肪滴直径および線維化面積においても、対照マウスと比較して、MEM マウス肝臓では双方とも増大していた。さらに、出生後の 10 週間の大麦投与により、肝線維化面積は減少、すなわち、肝線維化が抑制されたことが明らかとなった。そこで、肝線維化の重要なメディエーターである線維化促進因子 Tgfb の MEM マウス肝臓における mRNA 発現およびタンパク質発現を測定したところ、KSOM 対照マウスと比較して、MEM マウスの肝臓では Tgfb の mRNA およびタンパク発現が増大していることが示された。さらに、10週間大麦を投与された MEM マウスの肝臓では、この Tgfb 発現の増大が抑制されていることが明らかとなった。よって、胚環境操作モデル MEM マウスが発症する非アルコール性脂肪肝炎は肝線維化病態の軽減を伴って、大麦投与により抑制される可能性が示唆された。

第 3 章では、MEM マウスが糖尿病性腎症を呈するか、また出生後の大麦投与によりこの病態が抑制されうるかを検討した。その結果、MEM マウス腎臓糸球体では、対照マウスと比較して、線維化領域を指し示すメサンギウム領域が増大しており、それゆえメサンギウム領域の増大による糸球体結節性病変率が高いことが明らかとなった。さらに、出生後の 10 週間の大麦投与によりメサンギウム領域の増大および結節性病変率の上昇が抑制されていることが明らかとなった。また、糖尿病性腎症の特徴的な病態の一つである腎細動脈硬化を示す、硝子化様病変および内膜肥厚においても、KSOM 対照マウスと比較して、MEM マウスの腎臓では硝子化様病変率および内膜比が増大しており、 10 週間大麦を投与された MEM マウスの腎臓では、これらの病態は抑制されていることが示された。腎臓においても線維化メディエーターとして知られる Tgfb の遺伝子およびタンパク質の発現を測定したが、群間にて差は観察されなかった。一方で、腎臓は糸球体や尿細管など多様な細胞の集合体であるといった特徴を有していることから、 TGFB タンパク質の腎臓組織の免疫染色を行ったところ、MEM マウス糸球体ではTGFB発現の高まりは観察されなかったものの、近位あるいは遠位尿細管において、対照マウスと比較して、TGFB 発現領域が増大していることが明らかとなった。よって、胚環境操作モデル MEM マウスは、糸球体硬化症や細動脈硬化などを伴って糖尿病性腎症を発症し、中でも尿細管において病態進行している可能性が示唆された。加えて、出生後の大麦投与によりこれらの病態が抑制される可能性が示唆された。

以上の結果をまとめると、新規胚環境操作 2 型糖尿病マウスモデル MEM マウスは、日本人 2 型糖尿病患者と同様、軽度肥満にて食後高血糖を呈し、食後における末梢血白血球では、炎症性サイトカインやインテグリンなど炎症関連遺伝子の mRNA 発現が高まっていたことが明らかとなり、胚発生 2 細胞期におけるαMEM 培地の暴露並びに生後の高脂肪食の摂取は末梢血白血球の炎症関連遺伝子の発現が上昇することによって、 CVD 発症リスクを高めると想定された。さらに、高脂肪食を摂取した MEM マウスは肝臓における線維化病態や TGFB の高発現を伴って非アルコール性脂肪肝炎を呈することが明らかとなった。また、高脂肪食を摂取した MEM マウスの腎臓においては、糸球体硬化症や腎細動脈病変などの特徴を持っており、糖尿病性腎症を発症していることが示された。加えて、これらの病態は大麦投与により抑制されることが明らかとなった。よって日本人型 2 型糖尿病発症過程を探る研究、さらには、また病態予防のための食品因子の探索研究において、MEM マウスは適したモデルであることが示唆された。

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