JP2022530595A - Zc3h12bの遺伝子又はタンパク質の用途及び肝疾患動物モデルの確立方法 - Google Patents
Zc3h12bの遺伝子又はタンパク質の用途及び肝疾患動物モデルの確立方法 Download PDFInfo
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Abstract
Description
本発明の目的は、先行技術の不足に対して、ZC3H12Bの遺伝子又はタンパク質の用途を提供し、かつこれに基づいて肝疾患動物モデルの確立方法を提供することである。
候補物質で、ZC3H12B遺伝子又はタンパク質の発現系を処理することと、
前記系におけるZC3H12B遺伝子又はタンパク質の発現を検出することと、を含み、
前記候補物質がZC3H12B遺伝子又はタンパク質の発現を向上させることができる場合、その候補物質は必要な潜在物質であることを示し、この逆の場合は、候補物質は不要な潜在物質であることを示す。
本発明の好ましい実施形態として、前記肝疾患は肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん又はそれらに関連する脂肪肝又は肝臓がんから選択される。
本発明の他の好ましい実施形態として、前記動物は、ヒト以外の下等から高等脊椎動物が選択される。
a)ヒト及び/又は動物のzc3h12bによる、マクロファージが媒介する脂肪肝の発生及び肝臓がん様の変化の調節に関連する分子機構の研究と、
b)ヒト及び/又は動物の肝内胆管嚢胞腺腫、肝臓内胆管嚢胞腺がん又はそれらに関連する脂肪肝又は肝臓がんの分子発病機構の研究と、
c)肝内胆管嚢胞腺腫又は肝内胆管嚢胞腺がんの肝実質の萎縮壊死及び線維化がんの発病メカニズムと機構の研究と、
d)ヒトや動物において、雌雄の肝臓の分化又は発達に及ぼす影響、を及ぼす内因性の雌雄の性差により調節を受ける又は外因性の環境因子により調節を受ける肝内胆管嚢胞腺腫又は肝臓内胆管嚢胞腺がんの発生率への影響に関するスクリーニングと、から選択される。
本発明は、初めて遺伝子編集技術を利用して、メダカのzc3h12bの遺伝子を標的にノックアウトし、zc3h12bが欠失したメダカを樹立し、一連のフレームシフト・突然変異体、及び、Zc3h12bタンパク質生成が欠失したヘテロ接合体及びホモ接合体を作成した。これらのヘテロ接合体及びホモ接合体は、いずれも程度が異なる肝臓の病変を示した。月齢の増加に伴って、局所的な脂肪肝が現れ、胆管増生で線維化し、6か月齢のメダカでは著しい脂肪肝を示し、局部的に嚢腫して壊死していた。zc3h12bノックアウトメダカでは、正常の対照群に対して、肝類洞への顕著なリンパ球浸潤、マクロファージの数の異常な増加、肝胆管増生の融合と肝細胞脂肪病変が見られた。免疫組織化学反応では、平滑筋アクチンSMA陽性細胞だけでなく、GPC3、SMA、CK19などのヒトにおける肝臓および胆管腫瘍細胞マーカー陽性細胞も検出され、MMP9陽性マクロファージの数が有意に増加しており、鉄粒子の沈着などのヒトの肝臓がん様の症状を伴っていた。これに基づき:
1、zc3h12b欠失メダカは、肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん又はそれらの病変過程を研究する動物モデルとして、CCC3H型ジンクフィンガータンパク質であるZc3h12bによるマクロファージの炎症反応の活性化の制御、及び、肝臓以外の組織(生殖腺や脳など)固有のリポ多糖代謝-マクロファージ免疫応答についての分子機構を深く研究するための生体動物研究のプラットフォームを提供することができる。中医学(漢方)では、「肝臓がん」は「肥気」病と呼ばれていることから、zc3h12bノックアウトメダカは「肥気メダカ」と命名された。本発明の動物モデルは、従来技術において化学的に誘導した肝臓がん発生動物モデルと比べて、臨床的に肝内胆管嚢胞腺腫又は肝内胆管嚢胞腺がんの発生過程を良好にシミュレート(模擬)することができ、肝内胆管嚢胞腺腫及び肝内胆管嚢胞腺がんの遺伝子レベルの性差を深く探求することにも一層に役立ち、かつ単一遺伝子ノックアウトの方式は簡単で容易に操作できる。
2、ZC3H12B遺伝子又はタンパク質は、肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん、又はそれらに関連する脂肪肝又は肝臓がんの治療標的とすることができる。
3、ZC3H12B遺伝子又はタンパク質は、肝内胆管嚢胞腺腫、肝臓内胆管嚢胞腺がん又はそれらに関連する脂肪肝又は肝臓がんの診断マーカーとすることができる。
4、ZC3H12B遺伝子又はタンパク質を標的として、肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん、又はそれらに関連する脂肪肝又は肝臓がんを治療する薬物をスクリーニングすることができる。
図1:zc3h12bノックアウト型「肥気メダカ」:肝胆管増生の融合、肝細胞の風船様脂肪変性(***);肝類洞内リンパ球浸潤
Zc3h12b
Zc3h12bはZc3h12タンパク質ファミリーのメンバーであり、このタンパクファミリーは免疫応答と炎症反応に作用するCCCH型ジンクフィンガータンパク質である。
本発明において、使用されるZc3h12bタンパク質は天然に存在し得る。例えばそれは、下等から高等脊椎動物まで単離又は精製され得る。また、に分離又は純化され得る前記Zc3h12bタンパク質は、従来の遺伝子工学組換え技術に従って、組換えられたZc3h12bタンパク質を生産するように人工的に調製されてもよい。
本文で使用されるように、前記Zc3h12bのアップレギュレーターは、促進剤、作動薬などを含む。Zc3h12bタンパク質の活性を高め、Zc3h12bタンパク質の安定性を維持し、Zc3h12bタンパク質の発現を促進し、Zc3h12bタンパク質の分泌を促進し、Zc3h12bタンパク質の有効作用時間を延長し、又はZc3h12bの転写と翻訳を促進する物質はいずれも本発明に用いることができる。
本発明は、ZC3H12B遺伝子又はタンパク質、又はそれらのアップレギュレーターが、肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん、又はそれらに関連する脂肪肝又は肝臓がんを治療する薬物の調製における応用を提供する。
候補物質で、ZC3H12Bの遺伝子又はタンパク質の発現系を処理することと、
前記系におけるZC3H12Bの遺伝子又はタンパク質の発現を検出することと、を含み、
前記候補物質がZC3H12Bの遺伝子又はタンパク質の発現を向上させることができる場合、その候補物質は必要な潜在物質であることを示し、この逆の場合は、候補物質は不要な潜在物質であることを示す。
本発明の発見に基づいて、動物のzc3h12b遺伝子又はタンパク質発現をノックダウンするステップを含む肝疾患動物モデルの確立方法が提供される。前記肝疾患は肝臓内胆管嚢腫、肝内胆管嚢胞腺がん、又はそれらに関連する脂肪肝又は肝臓がんであることができる。動物はヒト以外の下等から高等脊椎動物に選ばれ、魚類、両生類、爬虫類、鳥類、及びネズミ、イヌ、ウサギ、サル、ヒトを含む哺乳類を含む。
本発明の薬物組成物は、本明細書に記載の活性剤及び薬学的に許容されるキャリアを含むことができる。薬学的に許容されるキャリアは通常、安全で無毒であり、広く製薬産業において薬物組成物を調製するためのいかなる公知物質を含むことができ、例えば、充填剤、希釈剤、凝固剤、粘着剤、潤滑剤、流動促進剤、安定化剤、着色剤、湿潤剤、崩壊剤などである。合成ペプチドの送達(デリバリー)に適した賦形剤を選ぶには、主にこの薬物組成物の投与方法を考慮必要があり、当業者はこの技術を熟知している。本発明の薬物における活性剤の含有量は、異なる治療用途に応じて決定することができる。上記の薬物組成物は、すでに知られている薬学プログラムによって調製できる。例えば、「レミトンの製薬科学」(Remington’s Pharmaceutical Sciences、第17版、Alfonoso R. Gennaro編、マック出版社(Mack Publishing Company)、イーストン、ペンシルバニア(1985))の本に詳細な記載がある。本発明の薬物は、カプセル剤、顆粒剤、錠剤、丸薬、内服液、注射液などを含むが、これらに限定されない。
1.zc3h12b遺伝子のノックアウト及び同定
CRISPR/Cas 9システムを利用して、メダカの1細胞期の受精卵をマイクロインジェクションし、zc3h12b遺伝子のノックアウトを行う。具体的な方法は次の通りである。
(Japanese medaka HdrR,Oryzias latipes)のzc3h12b遺伝子配列を取得する。Crisprscanのウェブサイト(http://crisprscan.org/)で、メダカのzc3h12b遺伝子配列に基づいて、CRISPR/CAS9編集システムの標的ノックアウト部位を検索するとともに、メダカのトランスクリプトームBLASTとの照合検索をすることで、他のいかなる非特異性的結合も予測されない候補部位を選択する。標的部位は、プロモーターの後に最初のATGに近い位置を選択する。標的シーケンスの前にT7プロモーターシーケンスを追加し、後端にgRNA scaffoldシーケンスを追加し、同時に、T7プロモーターの効率を確保するために、標的部位5’の端の最初の二つの塩基はGGであるべきであり、そうでなければCをGに変更する。本プロジェクトの実際の標的部位は以下の通りである。
zc3h12bCrispr1:GCATGCCACTGAGGAGTCGG(SEQ ID NO:1)
zc3h12bCrispr2:GGGAGAAACTAGGCCGGTCG(SEQ ID NO:2)
上海生工会社(Shanghai Biotech Co. Ltd.)によって合成され、合成シーケンスはそれぞれ以下の通りである。
(a)zc3h12bgRNA1:
5’-TAATACGACTCACTATAGGATGCCACTGAGGAGTCGGGTTTTAGAGCTAGAAATAGC(SEQ ID NO:3)
(b)zc3h12bgRNA2:
5’-TAATACGACTCACTATAGGGAGAAACTAGGCCGGTCGGTTTTAGAGCTAGAAATAGC(SEQ ID NO:4)
zc3h12bのgRNA1とzc3h12bのgRNA2は、それぞれPCR(94℃3分1ラウンド、94℃30秒、65℃30秒、72℃1分(34ラウンド)、72℃5 分1ラウンド)を介して、SgRNA-scaffold 5'-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC(SEQ ID NO:5)に連結した。PCR産物をQIAquick PCR Pruification Kitキットにより精製し、その後MAXIscript(登録商標)T7 In Vitro Transcription Kit(Thermo Fisher、USA)キットを用いてin vitroで転写し、gRNAを合成した。Cas 9タンパク質(金斯瑞生物科技、中国)と混合してマイクロインジェクションを行い、胚注射後の標的ノックアウト効率を検出し、最後に上記二つの標的部位に焦点を合わせる。
1) gRNA1(100ng/μl)、Cas9タンパク質(100ng/μl)添加;
2) gRNA1(100ng/μl)とgRNA2(100ng/μl)、Cas9タンパク質(100ng/μl)添加。
1)プライマー合成
Fw9:5’- GACTTAGACGGAGAAGACCATATTAG(SEQ ID NO:6)
Rv10:5’-CGCACCAATTCAGCAAGAAC(SEQ ID NO:7)
プライマーFw9とRv 10は、野生型とzc3h12bノックアウト型を識別するために、メダカのzc3h12bのエクソン1セグメント断片を特異的に増幅することができる。
メダカは26~28℃の水循環系で飼育され、明暗サイクル時間は14/10時間で、上海海洋大学実験動物研究委員会の指導に従って厳重に処理している。gRNA 1とgRNA 2の両方の標的を同時に注射し、遺伝子編集された成魚を種魚として、それぞれ野生型の雌雄とペアにして、発生した子を成魚に飼育し、尾びれを切ってDNAを抽出し、FW9とRV10プライマーを使ってPCRを行ってzc3h12b遺伝子型を同定する。PCR産物はpGEM(登録商標)-T Easy(Promega,USA)ベクターに接続され、プラスミドを抽出し、シークエンシングしてDNAシーケンスを同定した。確認されたヘテロ接合体の雌雄個体を互いにペアにし、子孫遺伝子型の同定によりメンデルの遺伝法則に合致することを示し、最終的にはzc3h12bノックアウトの編集をされたホモ接合体を得る。
性的に成熟したメダカ(3か月齢以上)の解剖と実体観察である。野生型の成魚、雌雄各3匹を準備する。ヘテロ接合体の雄2匹、ホモ接合体5匹で実体解剖を行い、4%パラホルムアルデヒド(PFA)を用いて肝臓、生殖腺などの組織を通常の方法で固定し、パラフィンに包埋し組織切片(厚さ6ミクロン)とし(Leica RM 2265自動ミクロトーム、ドイツ)、ヘマトキシリン・エオシン(HE)染色を施し、野生型個体及びzc3h12b遺伝子ノックアウト個体の肝臓などの組織学的形態変化を観察した。
鉄沈着マクロファージのプルシアンブルー染色方法:肝臓のパラフィン切片に対し、キシレンを用いて通常の脱蝋を行い、漸減濃度の一連のアルコールによって再水和し、脱イオン水で3回洗浄した後、新たに調製した20%塩酸+10%フェロシアン化カリウムを1:1で混合し、室温で光を避けて2時間染色し、4度で一晩放置した。翌日は30分室温に戻し、蒸留水で3回(5分/回)洗浄した後、エオシン染色を2~3分し、漸増濃度のアルコールで脱水し、キシレンに還元し、中性樹質で切片を封止した後、顕微鏡で観察し写真(ニコンNI-S-E)を撮影した。
Claims (10)
- ZC3H12b遺伝子又はタンパク質、又はそれらのアップレギュレーターが、肝内胆管嚢胞腺腫(biliary cystadenoma、BCA)、肝内胆管嚢胞腺がん(biliary cystadenocarcinoma、BCAC)、又はそれらに関連する脂肪肝又は肝臓がんを治療する薬物の調製における応用。
- 前記アップレギュレーターは、小分子化合物又は生体高分子から選択されることを特徴とする請求項1に記載の応用。
- 肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん、又はそれらに関連する脂肪肝又は肝臓がんを治療する薬物組成物であって、ZC3H12B遺伝子又はタンパク質のアップレギュレーター、及び薬学的に受け入れ可能なキャリアを含むことを特徴とする薬物組成物。
- ZC3H12B遺伝子又はタンパク質を診断マーカーとする、肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん、又はそれらに関連する脂肪肝又は肝臓がんの診断試薬又は診断キットの調製における応用。
- ZC3H12B遺伝子又は蛋白量を測定する試薬が、肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん、又はそれらに関連する脂肪肝又は肝臓がんの診断試薬又は試薬キットにおける応用。
- ZC3H12Bの遺伝子又はタンパクを標的として、肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん、又はそれらに関連する脂肪肝又は肝臓がんを治療する薬物をスクリーニングする方法であって、
候補物質で、ZC3H12B遺伝子又はタンパク質の発現系を処理することと、
前記系におけるZC3H12B遺伝子又はタンパク質の発現を検出することと、を含み、
前記候補物質がZC3H12B遺伝子又はタンパク質の発現を向上させることができる場合、その候補物質は必要な潜在物質であることを示し、この逆の場合は、候補物質は不要な潜在物質であることを示すことを特徴とする方法。 - 肝疾患動物モデルの確立方法において、動物のZC3H12B遺伝子又はタンパク質発現をノックダウンするステップを含むことを特徴とする確立方法。
- 前記肝疾患は、肝内胆管嚢胞腺腫、肝内胆管嚢胞腺がん、又はそれらに関連する脂肪肝又は肝臓がんであることを特徴とする請求項7に記載の応用。
- 前記動物は、ヒト以外の下等から高等脊椎動物から選択されることを特徴とする請求項7に記載の応用。
- 請求項7~9のいずれか1項に記載の方法に従って確立された肝疾患動物モデルの用途において、前記用途は、
a)ヒト及び/又は動物のzc3h12bによる、マクロファージが媒介する脂肪肝の発生及び肝臓がん様の変化の調節に関連する分子機構の研究と、
b)ヒト又は動物のZc3h12bによる肝臓内胆管嚢腺腫、肝臓内胆管嚢胞腺がん又はそれらに関連する脂肪肝又は肝臓がんの分子発病機構の研究と、
c)Zc3h12bによる肝内胆管嚢胞腺腫又は肝臓内胆管嚢胞腺がんの肝実質の萎縮壊死及び繊維化がんの発病性メカニズムと機構の研究と、
d)ヒトや動物において、雌雄の肝臓の分化又は発達に影響を及ぼし、肝内胆管嚢胞腺腫又は肝臓内胆管嚢胞腺がんの発生率に内在する雌雄性の差又は外因性環境因子の影響の鑑定スクリーニングと、から選択される用途であることを特徴とする。
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