JP2021116228A - 抗酸化ストレス剤、化粧品、および医薬品 - Google Patents
抗酸化ストレス剤、化粧品、および医薬品 Download PDFInfo
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Abstract
Description
(1) 間葉系幹細胞由来のExosomeを含む抗酸化ストレス剤、
(2) (1)に記載の抗酸化ストレス剤を配合させた化粧品、
(3) (1)に記載の抗酸化ストレス剤を配合させた酸化ストレスに対する予防効果および/または治療効果を有する医薬品
が提供される。
間葉系幹細胞は、骨細胞、心筋細胞、脂肪細胞、筋細胞、軟骨細胞など、間葉系に属する一種以上の細胞への分化能を有する多能性幹細胞である。また、間葉系幹細胞は、この分化能を保持したまま増殖することができる。また、間葉系幹細胞は、間葉系組織を有する全ての組織に存在すると考えられ、例えば、脂肪組織、臍帯、臍帯血、骨髄、胎盤、歯髄、羊膜、骨格筋、骨膜、子宮内膜等に間葉系幹細胞は存在する。本発明においては、間葉系幹細胞として、脂肪組織由来間葉系幹細胞、臍帯由来間葉系幹細胞、骨髄由来間葉系幹細胞、胎盤由来間葉系幹細胞、歯髄由来間葉系幹細胞等、特に限定することなく用いることができる。
また、自家細胞を用いる場合、自己間葉系細胞を用いてもよいし、他人の間葉系幹細胞を用いてもよい。
Exosomeの抽出方法としては、超遠心分離法、限外濾過法、ゲル濾過法、HPLC、抽出用試薬を用いる方法、Exosomeをトラップできるように処理されたろ紙を用いる方法等が挙げられる。
また、本発明の抗酸化ストレス剤は、肌細胞の保湿効果や抗加齢作用効果を有する。
また、本発明の抗酸化ストレス剤を配合させた食品や飲料を、錠剤、カプセル剤、シロップ等の経口投与製剤と同様の形態としてもよい。
ヒト脂肪組織由来間葉系幹細胞:Human Mesenchymal Stem Cells from Adipose Tissue (hMSC-AT)(C-12977)と専用培地(C-28009)及び、正常ヒト皮膚線維芽細胞(小児):Normal Human Dermal Fibroblasts (NHDF), juvenile foreskin(C-12300)と専用培地(C-23020)、はすべてPromo Cell社から購入して使用した。
過酸化水素水など生化学試薬は和光純薬工業株式会社より購入した。
間葉系幹細胞、線維芽細胞はそれぞれCulture Flaskを用いて、7日間培養し、80%程度コンフルエントになった状態の培養上清を回収し、遠心(12,000rpm、15分)によって細胞のデブリスを除去した上澄みをExosomeの単離まで4℃で保存した。
単離したExosomeは1/100量で(最終濃度;100pg/ml)培養液に添加して、4時間インキュベーションした。
0.2 mM過酸化水素水を添加した培地で、皮膚線維芽細胞を2時間インキュベーションしたのち、リン酸緩衝生理食塩水(PBS)で洗浄除去し、通常の培地に入れ替えてインキュベーションを継続した。その後、48時間経過後に細胞または培養液を回収し、細胞生存率の測定を行った。結果を図1のH2O2-NHDFとして示した。
上記「細胞への酸化ストレス処理及びExosome処理」における処理と同様の処理を行い、アクアポリン−1(AQP-1)およびアクアポリン−3(AQP-3)の量を測定し、結果をそれぞれ図2および図3に示した。アクアポリンの分泌刺激効果の検討については、皮膚繊維芽細胞に過酸化水素水を予め加えて、その後、間葉系幹細胞由来Exosomeを添加したもの(H2O2 / MSC-Exo.)、皮膚繊維芽細胞に過酸化水素水を予め加えて、その後、皮膚線維芽細胞由来Exosomeを加えたもの(H2O2 / NHDF-Exo.)、皮膚線維芽細胞に間葉系幹細胞由来Exosomeを添加し、その後、過酸化水素水を加えたもの(MSC-Exo. / H2O2)及び、皮膚線維芽細胞に皮膚線維芽細胞由来Exosomeを添加し、その後、過酸化水素水を加えたもの(NHDF-Exo. / H2O2)について測定を行った。また、皮膚線維芽細胞にExosomeも過酸化水素水も添加しないもの(Nontreat-NHDF)についても測定を行った。
なお、AQP-1および、AQP-3の測定としては、定量PCR法を用いた。
上記「細胞への酸化ストレス処理及びExosome処理」における処理と同様の処理を行い、ヒアルロン酸の量を測定し、結果を図4に示した。なお、測定は、細胞培養上清中のヒアルロン酸をELISA Kit , Hyaluronan Enzyme-Linked Immunosorbent Assay kit ( R&D systems.、 Inc. Mineapolis, MN 55413 USA)を用いて測定した。
上記「細胞への酸化ストレス処理及びExosome処理」における処理と同様の処理を行い、細胞内シグナル伝達にかかわる分子(SIRT1、P53及びP21)のmRNAレベルを測定し、結果をそれぞれ図5〜7に示した。また、老化細胞が染色される状態を顕微鏡下に観察し、蛍光量を数値化して、図8に示した。
上記「細胞への酸化ストレス処理及びExosome処理」における処理と同様の処理を行い、その後、それぞれの検体について、PBSで洗浄したのち、1M蛍光プローブ(CM-H2DCFDA (Molecular Probes Inc., Eugene, OR)を加え、37℃で60分インキュベーションした。そして、細胞内の活性酸素種(ROS)の産生量をfluorescence intensity using the micro plate reader (SYNERGY/HT, BioTek, Japan)を用いて測定し、蛍光量を数値化した。結果を図9に示した。
Claims (3)
- 間葉系幹細胞由来のExosomeを含む抗酸化ストレス剤。
- 請求項1に記載の抗酸化ストレス剤を配合させた化粧品。
- 請求項1に記載の抗酸化ストレス剤を配合させた酸化ストレスに対する予防効果および/または治療効果を有する医薬品。
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JP2022132330A JP7504411B2 (ja) | 2022-08-23 | ヒアルロン酸分泌刺激剤、ヒアルロン酸分泌刺激用化粧品、およびヒアルロン酸分泌刺激用医薬品 |
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