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大学・研究所にある論文を検索できる 「E3ユビキチンリガーゼTRIM27の機能と、鼻副鼻腔粘膜悪性黒色腫における発現の臨床病理学的解析に関する研究」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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E3ユビキチンリガーゼTRIM27の機能と、鼻副鼻腔粘膜悪性黒色腫における発現の臨床病理学的解析に関する研究

木村, 将吾 北海道大学

2023.06.30

概要

背景と目的
TRIM ファミリータンパク質は、細胞内シグナル伝達、発生、自然免疫、発がんな
どさまざまな分野において機能を有する。TRIM ファミリータンパク質の中でも
TRIM27 は悪性腫瘍の細胞増殖や遊走・浸潤能に影響を与えると報告されているが、
詳細なメカニズムについては未だ不明な点が多い。そこで、TRIM27 の機能を分子生
物学的に解析し、その分子メカニズムの解明を第一の目的とした。また、TRIM27 の
関与について未だ不明な点が多い頭頸部がんにおける TRIM27 発現と臨床経過の解明
を第二の目的とした。対象疾患として、近年、皮膚悪性黒色腫の臨床経過と TRIM27
発現との関与が報告されたことを踏まえ、頭頸部がんの1つである鼻副鼻腔粘膜悪性
黒色腫(sinonasal mucosal melanoma:SNMM)における TRIM27 発現と臨床経過について
解析を行った。
対象と方法
TRIM27 の悪性腫瘍における機能メカニズムを解明するために TRIM27 結合タンパク
質の同定を行った。レトロウイルスベクターを用いて FLAG-TRIM27 過剰発現細胞を樹
立し、抗 FLAG 抗体を用いた免疫沈降法により TRIM27 結合タンパク質を精製し質量分
析を行った。得られた結合タンパク質の結合と細胞内での局在をそれぞれウエスタン
ブロット法と蛍光免疫染色法によって解析した。mRNA の発現量は qPCR 法を用いて解
析した。また、タンパク質同士の相互作用を検証するために VIP assay (The visible
immunoprecipitation assay)を行った。VIP assay は結合を検討する 2 つのタンパク質
にそれぞれ GFP と mCherry タグを付加し、GFP 融合タンパク質を抗 GFP Nanobody を付
加したグルタチオンセファロースビーズで免疫沈降処理し、蛍光顕微鏡で mCherry の
蛍光を直接観察することで結合を評価した。
SNMM における TRIM27 の関与を解明するために 2003 年 5 月から 2021 年 3 月までに
治療を受けた 28 名の SNMM 患者を後方視的に検討した。SNMM 組織における TRIM27、
Ki-67、p-Akt1 の発現を免疫組織化学的に解析した。TRIM27 の発現と臨床的特徴、予
後、腫瘍増殖能マーカーである Ki-67、粘膜悪性黒色腫の予後因子である p-Akt1 との
関連性を検討した。
結果
FLAG-TRIM27 過剰発現 HEK293T 細胞を樹立し、抗 FLAG 抗体で免疫沈降後に質量分析
を行ったところ、BBS (Bardet-Biedl syndrome)1、BBS2、BBS4、BBS5、BBS8、BBS9 を結
合分子候補として同定した。Bardet-Biedl syndrome は先天奇形(多指症、合指症)

網膜色素変性、腎機能障害、肥満、精神発達遅滞などの症状をきたす遺伝性疾患であ
り、複数の責任遺伝子産物によって BBSome という複合体を形成する。 ...

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