JPWO2005094888A1 - 損傷組織の治療剤と治療方法 - Google Patents
損傷組織の治療剤と治療方法 Download PDFInfo
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Abstract
Description
内皮機能が回復することである。
(細胞培養)
MSCの分離は、Tsutsumiらの方法(Tsutsumi S、B.B.R.C. 2001;288:413-419)に従って行った。すなわち、4週齡の雄性日本白色ウサギ(SPF、北山ラベス生産)3羽の腹腔内へネンブタール麻酔薬を過剰投与して屠殺後、両側の大腿骨・脛骨を取り出し、骨端を落とし、この中に存在する骨髄を一本あたり約20mLのダルベッコ変法イーグル培地(シグマ社)(最終濃度100 unit/mLのペニシリンG、最終濃度100μg/mLの硫酸ストレプトマイシン、最終濃度0.0085%のアンホテリシンB(ギブコ社)および10%牛胎児血清を含む)で、21ゲージ針(テルモ社)を使用して骨髄を分離後、骨髄細胞の懸濁液を、ピペットを用いて単一の細胞にした。上述のダルベッコ変法イーグル培地を含む培養皿(175cm2、ファルコン社)に、片足分の大腿骨及び脛骨の骨髄液を全て播種し、底面に接着した細胞(MSC)をそのまま同じ培地で培養し続け(37℃、5% CO2 95% Air)、コロニーを形成したところで(7日目)、新たな培養皿に細胞密度5000細胞/cm2で継代培養した。
上記で得られたウサギ由来MSCに対する、PDGF−BB(Genzyme Techne社製)、bFGF(塩基性線維芽細胞成長因子)(科研製薬社製)、HB−EGF(シグマ社製)、TGF−α(DIACLONE Research社製)、PDGF−AB(Genzyme Techne社製)、IGF−I(Insulin-like growth factor-I)(BD Biosciences社製)、EGF(Pepro Tech EC社製)、α−トロンビン(Enzyme Research Laboratories社製)、HGF(Pepro Tech EC社製)、TGF−β1(Pepro Tech EC社製)、TGF−β3(Pepro Tech EC社製)、IL−2(Pepro Tech EC社製)、SCGF-α(Stem cell growth factor-α) (Pepro Tech EC社製)、SCF(Stem cell factor)(Pepro Tech EC社製)、SDF−1α(Stromal cell-defived factor-1α)(Pepro Tech EC社製)、レプチン(DIACLONE Research社製)、BDNF(Brain-derived neurotrophic factor)(和光純薬工業社製)、NGF−β(Nerve growth factor-β) (シグマ社製)、NT−3(Neurotrophin-3)(シグマ社製)、ANP(Biogenesis社製)、ヒアルロン酸(高分子、電気化学工業社製)、PDGF−AA(Pepro Tech EC社製)の各試験物質の遊走・集積刺激効果を検討した。
結果を図1,2のグラフに示す。縦軸はケモタキシスインデックス(CI)、すなわち、コントロールウェルのフィルター下部に移動した細胞の吸光度を基準として、試験物質を入れたウェルのフィルター下部に移動した細胞の吸光度を除した値を示す。統計学的検定はt検定で行った。図1の各グラフにおいて、*はp<0.05を表し、**はp<0.01を表す。図1,2の各グラフにおいて、バーは平均値±標準偏差(mean±SD)を表す。
ヒト腸骨由来のMSCである1F1061細胞および1F2155細胞(Cambrex社より購入)およびヒト顎骨由来のMSCであるKt−10細胞およびKt−11細胞を用いて、実施例1と同様の方法で細胞遊走試験を行った。
上記のヒト腸骨由来MSCおよびヒト顎骨由来MSCに対する、PDGF−BB、HB−EGF、TGF−α、PDGF AB、IGF−I、EGFの遊走・集積刺激効果を、実施例1と同じ方法で検討した。
実施例3
(細胞増殖試験)
実施例1で得られたウサギ由来MSCに対する、PDGF-BB(Genzyme Techne社製)、bFGF(塩基性線維芽細胞成長因子)(科研製薬社製)、HB−EGF(シグマ社製)、TGF−α(DIACLONE Research社製)、PDGF-AB(Genzyme Techne社製)、IGF−I(Insulin-like growth factor-I)(BD Biosciences社製)、EGF(Pepro Tech EC社製)、α−トロンビン(Enzyme Research Laboratories社製)、HGF(Pepro Tech EC社製)、PDGF−AA(Pepro Tech EC社製)、TGF−β1(Pepro Tech EC社製)、TGF−β3(Pepro Tech EC社製)、IL−2(Pepro Tech EC社製)、SCGF-α(Stem cell growth factor-α) (Pepro Tech EC社製)、SCF(Stem cell factor)(Pepro Tech EC社製)、SDF−1α(Stromal cell-defived factor-1α)(Pepro Tech EC社製)、レプチン(DIACLONE Research社製)、BDNF(Brain-derived neurotrophic factor)(和光純薬工業社製)、NGF-β(Nerve growth factor-β) (シグマ社製)、ANP(Biogenesis社製)の各試験物質の増殖刺激効果を検討した。
ウサギ由来MSCの遊走を促進させた因子(PDGF−BB、bFGF、HB−EGF、TGF−α、PDGF−AB、IGF−I、EGF、α−トロンビン、HGF)はすべてウサギ由来MSCの増殖を促進させた。一方、遊走に影響しなかった因子(PDGF−AA、TGF−β1、TGF−β3、IL−2、SCGF−α、SCF、SDF−1α、レプチン、BDNF、NGF−β、ANP)はウサギMSCの増殖を有意に促進しなかった。
実施例4
GFP(緑色蛍光タンパク質) トランスジェニックラットを使用して、MSC(GFP-MSC)のin vivo遊走試験を行った。
GFPトランスジェニックラットからのGFP-MSCの分離を行った。すなわち、腹腔内へネンブタール麻酔薬を過剰投与して屠殺後、両側の大腿骨・脛骨を取り出し、骨端を落とし、この中に存在する骨髄を一本あたり約20mLのダルベッコ変法イーグル培地(シグマ社)(最終濃度100 unit/mLのペニシリンG、最終濃度100μg/mLの硫酸ストレプトマイシン、最終濃度0.0085%のアンホテリシンB(ギブコ社)および10%牛胎児血清を含む)で、21ゲージ針(テルモ社)を使用して骨髄を分離後、骨髄細胞の懸濁液を、ピペットを用いて単一の細胞にした。上述のダルベッコ変法イーグル培地を含む培養皿(175cm2、ファルコン社)に、片足分の大腿骨及び脛骨の骨髄液を全て播種し、底面に接着した細胞(MSC)をそのまま同じ培地で培養し続け(37℃、5% CO2 95% Air)、コロニーを形成したところで(7日目)、新たな培養皿に細胞密度5000細胞/cm2で継代培養し、以下の実験に使用した。
ノントランスジェニックのSDラット(n=2)をネンブタールによって全身麻酔し(腹腔内40 mg/kg)、左右ふくらはぎ部を剃毛後、29G針を使用して、PDGF−BB 1μg/mlを含む2%アテロコラーゲン300μlを皮下注射した。その直後にGFP-MSC (2x106cells/ml、 150μl)を尾静脈へ注入した。コントロールにはPDGF−BBを含まない2%アテロコラーゲンのみを300μl皮下注射した。
7日後、コラーゲンを注入した部位を含むふくらはぎ部の筋肉および皮膚組織を採取し、組織中の細胞のtotal RNAを採取した。すなわちTRI Reagent(登録商標)(シグマ社)とRNeasy(登録商標)mini kit(QIAGEN社)によってtotal RNAを精製した。2μgの total RNAからOmniscript RT(QIAGEN社)を用いてcDNAを製作し、GAPDH(ハウスキーピング遺伝子) は1 μlのcDNA溶液を、GFPは4 μlのcDNA溶液をテンプレートとして、それぞれの特異的プライマーを使ってPCR(GAPDHは18 サイクル、GFPは40サイクル)を行った。得られたPCR産物は4% NuSieve(登録商標)GTG(登録商標) agarose ゲル(BMA社)内で電気泳動し、エチジウムブロマイド染色を行い、UV下で可視化した。GFPのPCR産物は129bp、GAPDHのPCR産物は613bpに対応するバンドとして現れる。
図5の電気泳動写真は、コラーゲン単独とPDGF−BB含有コラーゲンを注入したふくらはぎからそれぞれ採取したtotal RNAを用いてハウスキーピング遺伝子(GAPDH)とGFP遺伝子のRT-PCRを行った結果である。GFPに対応するバンドの存在は、尾静脈から注入したGFP-MSCがふくらはぎ部まで遊走したことを示す。GFPのRNA量についてコラーゲン単独を注入した側(PDGF-BB -)と、PDGF−BB含有コラーゲンを注入した側(PDGF-BB +)とで比較すると、個体1ではPDGF−BB含有側のみにGFPのバンドが認められ、個体2でもPDGF−BB含有側により強いバンドが認められた。これらのバンドをスキャンしてそれぞれのGAPDHのバンドの濃度を基準にしてGFPのバンドの濃度を補正したところ、個体1及び2のいずれにおいても、PDGF−BBを含有させた側のふくらはぎ部からより多くのGFP遺伝子が検出されることが示された(図6)。このことは、GFP-MSCが尾静脈から全身に循環する過程で、PDGF−BBが局在する部位へより多く遊走・集積したことを示す。
Claims (16)
- 損傷組織への間葉系幹細胞の遊走・集積を促進するおよび/または損傷組織からの間葉系幹細胞の拡散を抑制する薬剤または移植材。
- 間葉系幹細胞遊走能促進因子を含む請求項1記載の薬剤または移植材。
- 間葉系幹細胞の増殖を促進する間葉系幹細胞遊走能促進因子を含む、請求項2記載の薬剤または移植材。
- 再生治療に使用される請求項1〜3の何れか1項記載の薬剤または移植材。
- 変形性関節症、骨折、歯槽骨もしくは顎骨欠損、脳梗塞、心筋梗塞または下肢虚血による損傷組織の再生治療に使用される請求項4記載の薬剤または移植材。
- 間葉系幹細胞遊走能促進因子がEGF(上皮成長因子)、HB−EGF(ヘパリン結合上皮成長因子)、TGF−α、トロンビン、PDGF(血小板由来成長因子)、FGF(線維芽細胞成長因子)、ヒアルロン酸、IGF(インスリン様成長因子)、およびHGF(肝細胞増殖因子)から選ばれる請求項2〜5の何れか1項記載の薬剤または移植材。
- 間葉系幹細胞と同時に、連続的にまたは別個に投与される請求項1〜6の何れか1項記載の薬剤。
- 間葉系幹細胞と同時に、連続的にまたは別個に移植される請求項1〜6の何れか1項記載の移植材。
- 損傷組織への間葉系幹細胞の遊走・集積を促進することおよび損傷組織からの間葉系幹細胞の拡散を抑制することの少なくとも一方を含む損傷組織の再生治療方法。
- 間葉系幹細胞遊走能促進因子を投与することを含む請求項9記載の方法。
- 損傷組織が、変形性関節症、骨折、歯槽骨もしくは顎骨欠損、脳梗塞、心筋梗塞または下肢虚血による請求項9または10記載の方法。
- 間葉系幹細胞遊走能促進因子がEGF、HB−EGF、TGF−α、トロンビン、PDGF、FGF、ヒアルロン酸、IGF、およびHGFから選ばれる請求項10または11記載の方法。
- 間葉系幹細胞遊走能促進因子が損傷組織に局所投与される請求項10〜12の何れか1項記載の方法。
- 間葉系幹細胞遊走能促進因子が注射により投与される請求項13記載の方法。
- 間葉系幹細胞遊走能促進因子が損傷組織に塗布される請求項13記載の方法。
- 間葉系幹細胞遊走能促進因子の投与と同時に、連続的にまたは別個に、間葉系幹細胞を損傷組織および/またはその周辺に投与する請求項10〜15の何れか1項記載の方法。
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JP5852773B2 (ja) | 2010-06-01 | 2016-02-03 | ピアス株式会社 | 骨髄間葉系幹細胞の誘引剤の製造方法、骨髄間葉系幹細胞の誘引方法、及び、骨髄間葉系幹細胞の誘引剤を製造するための使用 |
US10195252B2 (en) | 2012-08-03 | 2019-02-05 | University of Pittsburgh—of the Commonwealth System of Higher Education | Recruitment of mensenchymal cells using controlled release systems |
US10507266B2 (en) | 2013-08-01 | 2019-12-17 | Two Cells Co., Ltd. | Cartilage-damage treatment agent and method for producing same |
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EP3708175A4 (en) * | 2017-11-09 | 2021-07-28 | Sapporo Medical University | MEDICINAL PRODUCT FOR TISSUE REGENERATION, AND ITS PREPARATION PROCESS |
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