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腸内常在菌が宿主粘膜恒常性に与える影響に関する基礎研究

大久 朋子 東北大学

2021.03.25

概要

目的
腸内常在細菌が腸粘膜上皮に重要な影響を及ぼすことが報告されている。本研究では、個々の細菌と消化管粘膜との直接的な作用をヒト腸上皮細胞の細胞間隙の変化やその機能遺伝子の転写変化、上皮細胞のムチン合成や代謝系の変化などを調べることで腸内細菌種間の粘膜機能に与える作用の違いを検討した。

材料及び方法
代表的な腸内常在細菌 49 株の培養上清中の有機酸を定量し、その種類・産生量から細菌株間クラスター解析を実施した。各クラスターから代表的な菌株、各々1 株以上を選択し、9 種類の被検菌株とした。ヒト結腸腺癌由来上皮細胞株(HT29 細胞)に細菌の培養上清または有機酸(酪酸、酢酸、乳酸)を曝露後に一定条件下で培養し、経上皮電気抵抗(TEER 法)による腸管粘膜のバリア機能である密着結合(TJs)を評価するとともに、TJs の関連遺伝子の転写を検討した。更に、腸粘膜に重要な作用を持つムチンの関連遺伝子の発現量を検討した。また、各培養上清または有機酸曝露培養後の HT29 細胞の代謝物を、ガスクロマトグラフィー質量分析法(GC/MS)を用いて測定することで細胞内代謝系の変化を評価した。

結果
供試細菌は有機酸産生パターンにより 7 種類のクラスターに分かれ、各クラスターから代表的な 9 株を選択し、培養上清を以降の実験に供した(L. plantarum、E. faecium、L. casei、F. nucleatum、B. longum、B. bifidum、B. fragilis、C. innocuum、B. ovatus)。各菌株または各有機酸を曝露した結果、非曝露(無添加)対照群と比較して、酪酸添加及び L. casei を除く 8 株の培養上清曝露により TEER 値が有意に上昇し(p<0.05)、粘膜バリア機能の向上が観察された。また、E. faecium、F. nucleatumの曝露により、TJs 関連遺伝子である CLDN-1の発現量が有意に増加した(p<0.05)。さらに、E. faecium、F. nucleatum、L. casei、B. ovatus、B. bifidum 及び C. innocuumにおいてムチン産生関連遺伝子の発現量が有意に増加した(p<0.05)。GC/MS を用いた HT29 細胞中の水溶性細胞内代謝物を網羅的に定性し、得られたデータを用いて解析を実施した結果、被験細菌は 5 つのクラスターに分類された。HT29 細胞の代謝物の網羅的検出により、22 種の代謝物が供試菌株の培養上清または有機酸の曝露によって検出濃度が有意に変動した(p<0.05)。特に、ムチン産生の代謝経路に関わる糖やアミノ酸が、一部の常在細菌の培養上清の曝露により上昇する傾向が認められ、菌種によって宿主上皮細胞に対する作用が異なることが示された。

結論
本研究の結果により、腸内細菌は、細菌単独でもその代謝産物を介して TJs やムチンの産生亢進などを示すが、その作用は菌株ごとに異なる特徴があることが示唆された。本研究手法は、in vitro で簡便に細菌―宿主腸管粘膜間の変化を観察できることから、粘膜破綻が関与するような状態、例えば TJs の破綻やムチン層の減弱による易感染状態を防御する細菌の探索への応用が期待される。

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