JP2011205964A - 腱及び/又は靭帯細胞の分化誘導剤 - Google Patents
腱及び/又は靭帯細胞の分化誘導剤 Download PDFInfo
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Abstract
【解決手段】腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化誘導剤は、(A)Mkxタンパク質、(B)前記Mkxタンパク質のアミノ酸配列において、1〜数個のアミノ酸が欠失、置換、逆位、付加及び挿入からなる群から選ばれる少なくとも一種の変異を有し、かつ、腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化を誘導する活性を有する変異体、(C)前記(A)又は(B)のタンパク質をコードする核酸、並びに、(D)前記(C)の核酸を含む発現ベクターの少なくとも一種を有効成分として含む。
【選択図】図1A
Description
(A)Mkxタンパク質、
(B)前記Mkxタンパク質のアミノ酸配列において、1〜数個のアミノ酸が欠失、置換、逆位、付加及び挿入からなる群から選ばれる少なくとも一種の変異を有し、かつ、腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化を誘導する活性を有する変異体、
(C)前記(A)又は(B)のタンパク質をコードする核酸、並びに、
(D)前記(C)の核酸を含む発現ベクター
の少なくとも一種を有効成分として含む、腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化誘導剤を提供する。
(A1)Scxタンパク質、
(B1)前記Scxタンパク質のアミノ酸配列において、1〜数個のアミノ酸が欠失、置換、逆位、付加及び挿入からなる群から選ばれる少なくとも一種の変異を有し、かつ、I型コラーゲンを誘導する活性を有する変異体、
(C1)前記(A1)又は(B1)のタンパク質をコードする核酸、並びに、
(D1)前記(C1)の核酸を含む発現ベクター、
の少なくとも一種をさらに含んでもよい。
(A)Mkxタンパク質、
(B)前記Mkxタンパク質のアミノ酸配列において、1〜数個のアミノ酸が欠失、置換、逆位、付加及び挿入からなる群から選ばれる少なくとも一種の変異を有し、かつ、腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化を誘導する活性を有する変異体、
(C)前記(A)又は(B)のタンパク質をコードする核酸、並びに、
(D)前記(C)の核酸を含む発現ベクター
(A1)Scxタンパク質、
(B1)前記Scxタンパク質のアミノ酸配列において、1〜数個のアミノ酸が欠失、置換、逆位、付加及び挿入からなる群から選ばれる少なくとも一種の変異を有し、かつ、I型コラーゲンを誘導する活性を有する変異体、
(C1)前記(A1)又は(B1)のタンパク質をコードする核酸、並びに
(D1)前記(C1)の核酸を含む発現ベクター。
1.Mkx遺伝子の機能的欠損を含む胚性幹細胞クローンを作製する工程、
2.上記胚性幹細胞クローンを胚にインジェクションする工程、
3.キメラ胚を動物の子宮に移して、キメラ動物を作る工程、
4.キメラ動物を交配させ、ヘテロ接合体を得る工程、及び
5.ヘテロ接合体同士を交配させ、ホモ接合体を得る工程、
を含む。
〔実施例1〕(分化誘導剤の作製と評価)
Mkxタンパク質をコードする核酸を含む発現ベクターからなる分化誘導剤を、C3H10T1/2マウス線維芽細胞に導入してMkxを過剰発現させることにより、分化誘導剤の効果を調べた。
マウスの染色体上のMkx遺伝子を機能的に欠損させるために、図1Aに、胚性細胞での相同組み換えにおいてMkxの翻訳スタートからエキソン2の終わりまでをMkx遺伝子がVenus遺伝子とネオマイシン耐性遺伝子とのカセットで置き換わるようなターゲッティングベクターを設計した。
実施例2で作製したMkxヘテロマウス同士を掛け合わせることにより、Mkxノックアウトマウスを作製した。産まれた各遺伝子型の新生仔マウスの数と比を表3に示す。
Claims (17)
- (A)Mkxタンパク質、
(B)前記Mkxタンパク質のアミノ酸配列において、1〜数個のアミノ酸が欠失、置換、逆位、付加及び挿入からなる群から選ばれる少なくとも一種の変異を有し、かつ、腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化を誘導する活性を有する変異体、
(C)前記(A)又は(B)のタンパク質をコードする核酸、並びに、
(D)前記(C)の核酸を含む発現ベクター
の少なくとも一種を有効成分として含む、腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化誘導剤。 - (A1)Scxタンパク質、
(B1)前記Scxタンパク質のアミノ酸配列において、1〜数個のアミノ酸が欠失、置換、逆位、付加及び挿入からなる群から選ばれる少なくとも一種の変異を有し、かつ、I型コラーゲンを誘導する活性を有する変異体、
(C1)前記(A1)又は(B1)のタンパク質をコードする核酸、並びに、
(D1)前記(C1)の核酸を含む発現ベクター、
の少なくとも一種を含む、請求項1に記載の腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化誘導剤。 - 腱及び/又は靭帯細胞への分化能を有する細胞の生育に適した条件下で、請求項1又は2に記載の分化誘導剤を用いて前記細胞を培養する工程、及び前記培養された細胞の中から、腱及び/又は靭帯細胞を選択する工程を含む、腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化を誘導する方法。
- 前記腱及び/又は靭帯細胞への分化能を有する細胞が、繊維芽細胞、腱前駆細胞、靱帯前駆細胞、胚性幹細胞、胚性生殖細胞、体性幹細胞及びiPS細胞からなる群から選ばれる少なくとも一種である、請求項3に記載の腱及び/又は靭帯細胞への分化能を有する細胞を腱及び/又は靭帯細胞に分化誘導する方法。
- 請求項1又は2に記載の腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化誘導剤を含む、腱又は靭帯関連疾患を予防又は治療するための医薬。
- 腱又は靭帯関連疾患が、エーラスダンロス症候群である、請求項5に記載の腱又は靭帯関連疾患を予防又は治療するための医薬。
- 請求項3又は4に記載の分化誘導方法により得られる腱及び/又は靭帯細胞を含む、腱又は靭帯関連疾患を予防又は治療するための医薬。
- Mkx遺伝子の機能的欠損を含む、腱又は靱帯関連疾患の研究用の遺伝子改変非ヒト動物。
- 前記機能的に欠損したMkx遺伝子は、Mkx遺伝子のプロモーターの下流にレポーター遺伝子が挿入されたことにより腱細胞特異的にレポーター遺伝子を発現する、請求項8に記載の腱又は靱帯関連疾患の研究用の遺伝子改変非ヒト動物。
- 前記レポーター遺伝子が、Venus遺伝子である、請求項9に記載の腱又は靱帯関連疾患の研究用の遺伝子改変非ヒト動物。
- 請求項1又は2に記載の分化誘導剤を用いることを特徴とする、腱又は靭帯関連疾患の解析又は分析方法。
- 請求項3又は4に記載の分化誘導方法により得られる腱及び/又は靭帯細胞を用いることを特徴とする、腱又は靭帯関連疾患の解析又は分析方法。
- 請求項8、9又は10に記載の遺伝子改変非ヒト動物の腱及び/靱帯、それらの組織又は細胞を用いることを特徴とする、腱又は靭帯関連疾患の解析又は分析方法。
- 被験物質の存在下又は非存在下で、請求項3又は4に記載の分化誘導方法を行い、該被験物質存在下又は非存在下での腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化を比較することを特徴とする、腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化調節に関連する物質をスクリーニング又は評価する方法。
- 被験物質の存在下で培養した細胞における、Mkxの発現量及び/又はMkxをコードする塩基配列に対するmRNAの転写量を測定すること、並びに前記被験物質を非存在下で培養した細胞内の量と比べて、前記測定した量が多い又は少ない場合に、前記被験物質を腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化調節に関連する物質として選択することを含む、請求項14に記載の腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化調節に関連する物質をスクリーニング又は評価する方法。
- 請求項8、9又は10記載の遺伝子改変非ヒト動物から得られる腱、その組織又はその細胞を用いることを特徴とする、腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化調節に関連する物質をスクリーニング又は評価する方法。
- 遺伝子改変非ヒト動物から得られる腱、その組織又はその細胞に被験物質を接触させること、及びノックインされたレポーター遺伝子の発現を上昇又は低下させる被験物質を選択することを含む、請求項16に記載の腱及び/又は靭帯細胞への分化能を有する細胞から腱及び/又は靭帯細胞への分化調節に関連する物質をスクリーニング又は評価する方法。
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CN106018574A (zh) * | 2016-05-05 | 2016-10-12 | 同济大学 | 对多肽或蛋白混合物进行简易快速离子交换分馏装置 |
WO2019013279A1 (ja) | 2017-07-13 | 2019-01-17 | 国立大学法人 東京医科歯科大学 | 三次元メカノシグナル細胞培養系を用いて作製した腱/靱帯様人工組織 |
US11773376B2 (en) | 2017-07-13 | 2023-10-03 | National University Corporation Tokyo Medical And Dental University | Artificial tendon or ligament tissue produced using three-dimensional mechanosignaling cell culture system |
WO2020235319A1 (ja) | 2019-05-20 | 2020-11-26 | 味の素株式会社 | 軟骨又は骨の前駆細胞の拡大培養方法 |
CN115094528A (zh) * | 2022-06-10 | 2022-09-23 | 东华大学 | 一种促进干细胞成肌腱分化的力学活性纤维及其制备方法 |
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