JP6185913B2 - ヒトの皮膚真皮由来成体幹細胞 - Google Patents
ヒトの皮膚真皮由来成体幹細胞 Download PDFInfo
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Description
正常ヒト皮膚線維芽細胞(Normal human dermal fibroblasts、NHDF)をロンザ社製(Lonza、Inc.Walkersville、MD、USA、NHDF−Ad−Der Fibroblasts、CC−2511)から購入した。前記ヒト皮膚線維芽細胞を75cm2T−フラスコに継代培養をし、CO2培養器(CO2 Incubator)で37℃、5%のCO2条件下で培養をした。通常、2回目または3回目で細胞の継代培養(passage)を行った。
ゼラチンまたはIV型コラーゲンで分離されたRA/SA細胞の幹細胞能(stemness)を比較するために、コロニー形成アッセイを確認した。
実施例1で既述のとおり、分離したヒト真皮由来成体幹細胞及びヒト皮膚線維芽細胞とを2mlのPBSにて洗浄した後、次いで、トリゾール試薬(Trizol reagent、Invitrogen、Carlsbad、CA、USA)を用いて細胞内のRNAを分離した。分離されたRNAをキアゲン社製のRNAキット(QIAGEN RNeasy kit、QIAGEN、Valencia、CA)でさらに一回精製した後、アジレント・テクノロジー社製のAgilent 2100バイオアナライザー(Agilent 2100 BioAnalyzer、Agilent Technologies、Santa Clara、CA、USA)を用いてRNAの質(quality)を確認した。インビトロジェン社製の逆転写キット(Superscript Reverse Transcriptase(RT) kit、Invitrogen、Carlsbad、CA)を用いて前記RNAからcDNAを合成し、これをリアルタイム逆転写重合酵素連鎖反応(real time−reverse transcription polymerase chain reaction、Q−RT−PCR)により定量的に分析した。
ゼラチンまたはIV型コラーゲンで分離したヒト真皮由来成体幹細胞の分化能を把握するために、骨芽細胞(Osteoblast)と脂肪細胞(Adipocyte)とにそれぞれ分化を誘導した。
分離したヒト真皮由来成体幹細胞及びヒト皮膚線維芽細胞を骨芽細胞と脂肪細胞にそれぞれ分化を誘導した後、前記実施例3と同じ方法にて、骨芽細胞のバイオマーカーであるOGN、ACANと脂肪細胞のバイオマーカーであるPPARG、Leptin、AdipoQ、FABP4遺伝子の発現パターンの変化をアプライドバイオシステム社のタックマン遺伝子発現システム(TaqMan gene expression assay kit、Applied Biosystems、Foster City、CA)(OGN遺伝子プライマー:Hs00247901_m1、ACAN遺伝子プライマー:Hs00153936_m1、PPARG遺伝子プライマー:Hs01115513_m1、Leptin遺伝子プライマー:Hs00174877_m1、AdipoQ遺伝子プライマー:Hs00605917_m1、FABP4遺伝子プライマー:Hs01086177_m1)を用いて評価し、その結果を図3及び図4に示した。分離したヒト真皮由来成体幹細胞の各遺伝子の発現量が、ヒト皮膚線維芽細胞に比べて、約2倍以上も増加したことを確認した。このような増加は、統計的に有意であり、統計は両側スチューデントのt検定にて検証し、p値が0.05以下のときに有意な差異があると表示した。
分離したヒト真皮由来成体幹細胞から培養液に分泌する成長因子の発現様相を把握するために、成長因子アレイ(Growth factor array、Raybio、Norcross、GA、USA)を実施した。
Claims (17)
- ヒト皮膚線維芽細胞に比べてS100b(S100 calcium binding protein B)遺伝子、またはSox2(SRY (sex determining region Y)−box 2)遺伝子及びS100b遺伝子が過発現する、寄託番号KCTC11995BPの幹細胞であることを特徴とするヒトの皮膚真皮由来成体幹細胞。
- 前記幹細胞は、
ヒト皮膚線維芽細胞を継代培養してからゼラチンまたはIV型コラーゲンに反応させ、ゼラチンまたはIV型コラーゲンに付着する細胞を分離して得られたものであることを特徴とする請求項1に記載のヒトの皮膚真皮由来成体幹細胞。 - 前記幹細胞は、
前記線維芽細胞を1〜5分間ゼラチンまたはIV型コラーゲンに反応させて得られたものであることを特徴とする請求項2に記載のヒトの皮膚真皮由来成体幹細胞。 - 前記ゼラチンは、蒸留水に0.1〜1質量%の濃度で溶解させてなるものであり、また前記IV型コラーゲンは、蒸留水に10〜30μg/mlの含量で溶解させてなるものであることを特徴とする請求項2に記載のヒトの皮膚真皮由来成体幹細胞。
- 前記ゼラチンまたはIV型コラーゲンは、0〜10℃の温度で16〜24時間基質にコーティングさせてなるものであることを特徴とする請求項2に記載のヒトの皮膚真皮由来成体幹細胞。
- 前記幹細胞は、
ヒト皮膚線維芽細胞に比べてEGF(Epidermal growth factor)、FGF4(Fibroblast growth factor4)、PDGF−AA(Platelet−derived growth factor−AA)、VEGF R2(Vascular endothelial growth factor receptor 2)、VEGF R3(Vascular endothelial growth factor receptor 3)及びVEGF D(Vascular endothelial growth factor D)からなる群より選ばれる一つ以上の成長因子が過発現することを特徴とする請求項1に記載のヒトの皮膚真皮由来成体幹細胞。 - 請求項1〜6の何れかに記載のヒトの皮膚真皮由来成体幹細胞を有効成分として含む骨生成用または脂肪生成用組成物。
- 前記組成物は、
骨粗鬆症の予防及び治癒、皮膚老化の予防及び治癒、皮膚傷の治癒、皮膚血行の改善、皮膚のボリューム感の増進、または皮膚成形効能を有することを特徴とする請求項7に記載の骨生成用または脂肪生成用組成物。 - ヒト皮膚線維芽細胞を継代培養してからゼラチンまたはIV型コラーゲンに反応させ、ゼラチンまたはIV型コラーゲンに付着する細胞を分離することを含むヒトの皮膚真皮由来成体幹細胞の分離方法。
- 前記分離方法では、
前記線維芽細胞を1〜5分間ゼラチンまたはIV型コラーゲンに反応させることを特徴とする請求項9に記載のヒトの皮膚真皮由来成体幹細胞の分離方法。 - 前記分離方法では、
前記ゼラチンを蒸留水に0.1〜1質量%の濃度で溶解させ、また前記IV型コラーゲンを蒸留水に10〜30μg/mlの含量で溶解させて線維芽細胞に反応させることを特徴とする請求項9に記載のヒトの皮膚真皮由来成体幹細胞の分離方法。 - 前記分離方法では、
前記ゼラチンまたはIV型コラーゲンを0〜10℃の温度で16〜24時間基質にコーティングして線維芽細胞に反応させることを特徴とする請求項10に記載のヒトの皮膚真皮由来成体幹細胞の分離方法。 - 前記分離方法は、
ヒト皮膚線維芽細胞に比べてS100b(S100 calcium binding protein B)遺伝子、またはSox2(SRY (sex determining region Y)−box 2)遺伝子及びS100b遺伝子を過発現するか否かを確認することをさらに含む請求項9に記載のヒトの皮膚真皮由来成体幹細胞の分離方法。 - 前記分離方法は、
ヒト皮膚線維芽細胞に比べてEGF(Epidermal growth factor)、FGF4(Fibroblast growth factor4)、PDGF−AA(Platelet−derived growth factor−AA)、VEGF R2(Vascular endothelial growth factor receptor 2)、VEGF R3(Vascular endothelial growth factor receptor 3)及びVEGF D(Vascular endothelial growth factor D)からなる群より選ばれる一つ以上の成長因子を過発現するか否かを確認することを含む請求項9に記載のヒトの皮膚真皮由来成体幹細胞の分離方法。 - ヒトの皮膚真皮由来成体幹細胞の分化方法であって、
請求項9〜14の何れかに1項に記載の分離方法によりヒトの皮膚真皮由来成体幹細胞を分離し、該幹細胞を骨芽細胞または脂肪細胞に分化させることを含み、
前記幹細胞は、寄託番号KCTC11995BPの幹細胞であり、
前記幹細胞は、ヒト皮膚線維芽細胞に比べてS100b遺伝子が過発現するか、又はSox2遺伝子及びS100b遺伝子が過発現している、
分化方法。 - 前記分化方法は、
分離された幹細胞を骨細胞分化培地で分化させた後、
分化された細胞がヒト皮膚線維芽細胞に比べてOGN(Osteoglycin)及びACAN(Aggrecan)からなる群より選ばれる一つ以上の遺伝子を過発現するか否かを確認することを含む請求項15に記載のヒトの皮膚真皮由来成体幹細胞の分化方法。 - 前記分化方法は、
分離された細胞を脂肪細胞分化培地で分化させた後、
分化された細胞がヒト皮膚線維芽細胞に比べてPPARG(Peroxisome proliferator−activated receptor gamma)、LEP(Leptin)、AdipoQ(Adiponectin、C1Q and collagen domain containing)及びFABP4(Fatty acid binding protein 4、adipocyte)からなる群より選ばれる一つ以上の遺伝子を過発現するか否かを確認することを含む請求項16に記載のヒトの皮膚真皮由来成体幹細胞の分化方法。
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KR20160022667A (ko) | 2014-08-20 | 2016-03-02 | (주)아모레퍼시픽 | 인간 진피 유래 성체 줄기세포를 유효성분으로 함유하는 혈관 생성 촉진용 조성물 |
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EP3091084A1 (en) | 2015-05-08 | 2016-11-09 | Université Catholique De Louvain | Methods for assessing the purity of a mesenchymal stem cells preparation |
KR102254600B1 (ko) * | 2020-02-13 | 2021-05-21 | 주식회사 피씨지바이오 | 간세포 회수율이 향상된 상피세포 분리방법 |
KR20220154429A (ko) | 2021-05-13 | 2022-11-22 | 건국대학교 글로컬산학협력단 | 분꽃(Mirabilis jalapa), 단풍(Acer palmatum) 추출물 등을 포함하는 탈모 예방 및 두피 상태 개선용 설페이트 프리 샴푸 조성물 |
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US5436135A (en) * | 1985-09-02 | 1995-07-25 | Pasteur Merieux Serums Et Vaccins | New preparation of placenta collagen, their extraction method and their applications |
AU5743601A (en) * | 2000-04-28 | 2001-11-12 | Childrens Medical Center | Isolation of mesenchymal stem cells and use thereof |
KR20060016540A (ko) * | 2004-08-18 | 2006-02-22 | 박경찬 | 피부 성체줄기세포 분리방법 |
KR100825864B1 (ko) | 2006-09-21 | 2008-04-28 | 부산대학교 산학협력단 | 피부 유래 줄기세포를 함유하는 신경질환 치료용세포치료제 |
CA2723848C (en) * | 2008-05-09 | 2013-01-08 | Regenics As | Method of making salmon egg cytoplasmic extracts and use for increasing collagen production in skin |
KR20100067277A (ko) * | 2008-12-11 | 2010-06-21 | 피더블유제네틱스코리아 주식회사 | 포유동물 피부유래 줄기세포를 골세포로 분화시키는 방법 |
US9550975B2 (en) * | 2009-07-15 | 2017-01-24 | Mari Dezawa | SSEA-3 pluripotent stem cell isolated from body tissue |
US8962323B2 (en) * | 2009-09-15 | 2015-02-24 | Shiseido Company, Ltd. | Method of isolating dermal stem cells |
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CN103827294B (zh) | 2017-07-21 |
KR101751355B1 (ko) | 2017-07-04 |
KR20130022484A (ko) | 2013-03-07 |
US20140170122A1 (en) | 2014-06-19 |
CN103827294A (zh) | 2014-05-28 |
US20170114326A1 (en) | 2017-04-27 |
US10865386B2 (en) | 2020-12-15 |
JP2014528710A (ja) | 2014-10-30 |
WO2013028034A1 (ko) | 2013-02-28 |
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