JP5900921B2 - 細胞増殖抑制剤、細胞または臓器の保存液 - Google Patents
細胞増殖抑制剤、細胞または臓器の保存液 Download PDFInfo
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- JP5900921B2 JP5900921B2 JP2012053798A JP2012053798A JP5900921B2 JP 5900921 B2 JP5900921 B2 JP 5900921B2 JP 2012053798 A JP2012053798 A JP 2012053798A JP 2012053798 A JP2012053798 A JP 2012053798A JP 5900921 B2 JP5900921 B2 JP 5900921B2
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
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- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Description
<1>次式で表される化合物。
<3>この化合物では、アシル基の炭素数が12〜20であることがより好ましい。
<4>この化合物では、アシル基の炭素数が16であることがさらに好ましい。
<5>前記化合物を有効成分として含む細胞増殖抑制剤。
<6>前記化合物を有効成分として含む細胞または臓器の保存液。
<7>この保存液では、有効成分の濃度が0.1μM〜1000μMであることが好ましい。
<8>前記の保存液に、細胞または臓器を浸漬することを特徴とする細胞または臓器の保存方法。
<9>この細胞または臓器の保存方法では、保存液に有効成分として含有される化合物は、アシル基の炭素数が6から28であることが好ましい。
<10>この細胞または臓器の保存方法では、保存液の有効成分の濃度が0.1μM〜1000μMであることが好ましい。
<11>この細胞または臓器の保存方法では、保存液の温度は、5〜37℃であることが好ましい。
<12>前記の細胞増殖抑制剤を添加した培地で細胞を培養することを特徴とする細胞培養方法。
ペプチド合成装置(PSSM−8、(株)島津製作所製)を用いて、通常の方法によって樹脂上にN末端遊離、保護基(NH2)付ペプチド:アスパラギン−イソロイシン−プロリン−アルギニン−ロイシン−NH2(NIPRL−NH2)を合成した。そして、上記ペプチド樹脂をジメチルホルムアミド(DMF)とピリジンの混合溶媒に懸濁し、パルミチン酸(C16カルボン酸)およびWSCD(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride)を加え、室温で一晩攪拌した。反応終了後、樹脂を濾集し、DMFおよびメタノールで洗浄した。このようにして得たアシル化ペプチド樹脂を通常の方法で切り落としカクテル処理し、N末端に炭素数が16のアシル基(パルミトイル基)を有する粗N末端アシル化NIPRL−NH2(C16−NIPRL−NH2)を得た。
(1)Penetratin−DILRG−NH2:「RQIKIWFQNRRMKWKK」のアミノ酸16残基配列を有するペプチドのC末端をDILRG−NH2のN末端に酸アミド結合させた化合物
(2)Oligoarginine−DILRG−NH2:「RRRRRRRRRR」のアミノ酸10残基配列を有するペプチドC末端をDILRG−NH2のN末端に酸アミド結合させた化合物
〈参考文献:生化学(第81巻、第11号、992-995、中瀬生彦、二木史朗 (京都大学化学研究所生体機能設計化学研究領域))〉
(3)AHPN−DILRG−NH2およびAHPN−FGPRL−NH2:AHPN(6−(3−(1−Adamantyl)−4−hydroxyphenyl)−2−naphtalene carboxylic acid)をDILRG−NH2またはFGPRL−NH2のN末端に酸アミド結合させた化合物
(1)K562(CML、慢性骨髄性白血病)
培地はRPMI1640(10%FBS 添加)を用い、1×105Cell/mL の濃度のK562細胞液を96wellのマイクロプレートに100μL/wellで捲き込んだ後、ペプチド誘導体各種につき、ヤママリン(DILRG−NH2)はリン酸バッファーに、その他のペプチド誘導体はDMSOに溶解し、それぞれの終濃度(以下の表1に記載)になるように調整後、各wellにヤママリン(DILRG−NH2)は10μL、その他のペプチド誘導体は0.5μLを添加した。37℃、5%CO2の条件下で、48時間培養した後、Premix WST-1 試薬(タカラバイオ株式会社製)を用いて、細胞数を測定した。すなわち、Premix WST-1試薬を各wellに試薬10μLを直接添加後、37℃、5%CO2の条件下で1時間反応させ、450/620nmで吸光度を測定した。
培地はDMEM(10%FBS 添加)を用い、1×105Cell/mL の濃度のHepG2細胞液を96wellのマイクロプレートに100μL/wellで捲き込み、37℃、5%CO2条件下で24時間培養後、ペプチド誘導体各種につき、ヤママリン(DILRG−NH2)はリン酸バッファーに、その他のペプチド誘導体はDMSOに溶解し、それぞれの終濃度(以下の表1に記載)になるように調整後、各wellにヤママリン(DILRG−NH2)は10μL、その他のペプチド誘導体は0.5μLを添加した。37℃、5%CO2の条件下で、48時間培養した後、Premix WST-1 試薬(タカラバイオ(株))を用いて、細胞数を測定した。すなわち、Premix WST-1試薬を各wellに試薬10μLを直接添加後、37℃、5%CO2の条件下で1時間反応させ、450/620nmで吸光度を測定した。
培地はNHDF専用培地(LIFELINE社製)を用い、0.8 ×104Cell/mLあるいは2.5×104cell/mL の濃度のNHDF細胞液を96wellのマイクロプレートに100μL/wellで捲き込み、37℃、5%CO2条件下で24時間培養後、ペプチド誘導体各種につき、ヤママリン(DILRG−NH2)はリン酸バッファーに、その他のペプチド誘導体はDMSOに溶解し、それぞれの終濃度(以下の表1に記載)になるように調整後、各wellにはヤママリン(DILRG−NH2)10μL、その他のペプチド誘導体は0.5μLを添加した。37℃、5%CO2の条件下で、48時間培養した後、Premix WST-1 試薬(タカラバイオ(株)製)を用いて、細胞数を測定した。すなわち、Premix WST-1試薬を各wellに試薬10μLを直接添加後、37℃、5%CO2の条件下で2時間反応させ、450/620nmで吸光度を測定した。
前記の方法により行った細胞増殖抑制試験の結果を表1に示す。細胞増殖抑制効果については、以下のように評価した。◎:高い効果を示した、○:効果を示した、△:やや効果を示した、×:効果は認められなかった、−:未実施
(1)細胞増殖抑制試験の組織培養方法及び測定方法
培地はRPMI1640(10%FBS 添加)を用い、1×105Cell/mL の濃度のK562細胞液を96wellのマイクロプレートに100μL/wellで捲き込んだ後、C16−NIPRL−NH2をDMSOに溶解し、それぞれの終濃度(0.1μM、1μM、10μM、25μM)になるように調整後、各wellに0.5μL添加した。37℃、5%CO2の条件下で、1、2、3、4、5日後培養した後、Premix WST-1 試薬(タカラバイオ(株)製)を用いて、細胞数を測定した。すなわち、Premix WST-1試薬を各wellに試薬10μLを直接添加後、37℃、5%CO2の条件下で1時間反応させ、450/620nmで吸光度を測定した。また、DILRG−NH2(ヤママリン)10mM、C16−DILRG−NH2(C16−ヤママリン)25μM、C16−NIPRL−NH2:0.1μM、1μM、10μM、25μM(いずれも終濃度)添加3日後のK562細胞に対する細胞増殖制御効果を比較した。
前記の方法により行った細胞増殖抑制試験の結果を図1、図2に示す。
図1は、K562細胞に対するC16−NIPRL−NH2の添加濃度と培養日数による細胞増殖抑制効果の変化を確認した図である。
K562細胞に対するC16−NIPRL−NH2の添加濃度と培養日数による細胞増殖抑制効果の変化を観察したところ、C16−NIPRL−NH2は添加後3日目では、濃度1μMで数%、10μMで50%、25μMで75%程度細胞増殖抑制効果を示すことがわかった。また添加濃度25μMでは、添加初日から細胞増殖抑制効果が顕著に現れ、少なくとも3、4日間は効果が持続することがわかった(図1)。
C16−NIPRL−NH2は、C16−DILRG−NH2(C16−ヤママリン)と使用濃度が同じ場合(25μM)、約2倍の細胞増殖抑制効果を示した。また、図2中では、C16−NIPRL−NH2の細胞増殖抑制効果は、DILRG−NH2(ヤママリン)の約2割増として示されているが、ヤママリンの使用濃度が10mMであることから換算すると、実際は、約480倍の効果を有すると考えられる。以上より、C16−NIPRL−NH2は、DILRG−NH2(ヤママリン)、C16−DILRG−NH2(C16−ヤママリン)に比べ、低濃度で細胞増殖抑制作用を示すことが判明した(図2)。
DILRG−NH2(ヤママリン)、C16−DILRG−NH2(C16−ヤママリン)、C16−NIPRL−NH2の、通常使用される培地各種への溶解性を調べた。その結果を表2に示す。なお、NHDF専用培地は、LIFRLINE社製のものを使用した。
Claims (12)
- アシル基の炭素数が6〜28である請求項1の化合物。
- アシル基の炭素数が12〜20である請求項1の化合物。
- アシル基の炭素数が16である請求項1の化合物。
- 請求項1から4のいずれかの化合物を有効成分として含む細胞増殖抑制剤。
- 請求項1から4のいずれかの化合物を有効成分として含む細胞または臓器の保存液。
- 有効成分の濃度が0.1μM〜1000μMであることを特徴とする請求項6の保存液。
- 請求項6の保存液に、細胞または臓器を浸漬することを特徴とする細胞または臓器の保存方法。
- 保存液に有効成分として含有される化合物は、アシル基の炭素数が6〜28であることを特徴とする請求項8の細胞または臓器の保存方法。
- 保存液の有効成分の濃度が0.1μM〜1000μMであることを特徴とする請求項8または9の細胞または臓器の保存方法。
- 保存液の温度は、5〜37℃であることを特徴とする請求項8から10のいずれかの細胞または臓器の保存方法。
- 請求項5の細胞増殖抑制剤を添加した培地で細胞を培養することを特徴とする細胞培養方法。
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