JP4462706B2 - Hematopoietic stem cell proliferating agent containing macrophage migration inhibitory factor - Google Patents
Hematopoietic stem cell proliferating agent containing macrophage migration inhibitory factor Download PDFInfo
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- JP4462706B2 JP4462706B2 JP2000096379A JP2000096379A JP4462706B2 JP 4462706 B2 JP4462706 B2 JP 4462706B2 JP 2000096379 A JP2000096379 A JP 2000096379A JP 2000096379 A JP2000096379 A JP 2000096379A JP 4462706 B2 JP4462706 B2 JP 4462706B2
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Description
【０００１】
【発明の属する技術分野】
本発明は、造血幹細胞の生存維持活性、増殖活性、および分化活性を有する造血幹細胞増殖因子としてのマクロファージ遊走阻止因子を含有し、各種の造血器官疾患、癌の放射線治療および化学療法の際の造血不全に対する治療薬として利用され得る造血幹細胞増殖剤に関する。本発明の造血幹細胞増殖剤はまた、血球の大量生産、および遺伝子治療時の造血幹細胞への遺伝子の導入効率向上に利用され得、さらに診断薬および検査薬として活用され得る。
【０００２】
【従来の技術】
造血幹細胞はすべての成熟した血球に分化する能力（多分化能）と、自己と同じ能力を有する細胞を複製する能力（自己複製能）を有し、長期に亘り造血を支持し続けている。ヒトの生体における造血は、一般に、細胞間の直接的な相互作用および体液中に存在する種々の物質（すなわち、液性の造血調節因子）により、調節されていると考えられる。細胞間の直接的な相互作用としては、造血幹細胞、特定の血球に分化することがコミットされた造血前駆細胞、およびこれらを取り巻く造血微小環境である間質細胞の間の相互作用が挙げられる。
【０００３】
液性の造血調節因子は、ここ十数年のバイオテクノロジーの進歩により、種々のものが見出された。例えば、エリスロポエチン（リンら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．、８２：７５８０（１９８５））、顆粒球コロニー刺激因子（ナガタら、ＥＭＢＯ Ｊ、５：５７５（１９８６））、顆粒球マクロファージコロニー刺激因子（ミヤタケら、ＥＭＢＯ Ｊ、４：２５６１（１９８５））、マクロファージコロニー刺激因子（ウオングら、Ｓｃｉｅｎｃｅ、２３５：１５０４（１９８７））、トロンボポエチン（ソーベッジら、Ｎａｔｕｒｅ、３６９：５３３（１９９４））、幹細胞成長因子（アンダーソンら、Ｃｅｌｌ、６３：２３５（１９９０））、Ｆｍｓ様チロシンキナーゼ３リガンド（リマネットら、Ｃｅｌｌ、７５：１１５７（１９９３））、白血病抑制因子（スタールら、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．、２６５、（１５）：８８３３（１９９０））、インターロイキン１（クラークら、Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．、１４、：７８９７（１９８６））、インターロイキン３（ドルサーズら、Ｇｅｎｅ、５５：１１５（１９８７））、インターロイキン６（ヤスカワら、ＥＭＢＯ Ｊ、６：２９３９（１９８７））、インターロイキン１１（ポールら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．、８７：７５１２（１９９０））、インターロイキン１２（ウオルフら、Ｊ．Ｉｍｍｕｎｏｌ．１４６：３０７４（１９９１））などである。これらのサイトカインはいずれも、造血幹細胞を、単独ではあまり増殖させることができず、ある程度増殖させるためには2つ以上、好ましくは3つ以上を組み合わせて用いることが必要である。従って、造血幹細胞をより効率的に増殖させ得る血球増殖因子を提供することが望まれている。
【０００４】
マクロファージ遊走阻止因子は、テンジクネズミのマクロファージの遊走を阻害するタンパク質として、最初に見出されたリンフォカインである（ブルームら、Ｓｃｉｅｎｃｅ、１５３：８０（１９６６）；デービットら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．、５６：７２（１９６６））。また、マクロファージ遊走阻止因子活性の発現は、動物モデルやヒトにおいて、遅延型過敏症や細胞免疫に関連している（ブルームら、Ｓｃｉｅｎｃｅ、１５３：８０（１９６６）；デービットら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．、５６：７２（１９６６）；デービットら、Ｐｒｏｇ．Ａｌｌｅｒｇｙ 、１６：３００（１９７２）；ロックリンら、Ｎ．Ｅｎｇｌ．Ｊ．Ｍｅｄ．、２８２：１３４０（１９７０））。さらに、マクロファージ遊走阻止因子活性は、マウスの他家移植片の拒絶時に、白血球の培養上清に検出されるし（アルアスカリら、Ｎａｔｕｒｅ、２０５：９１６（１９６５）；ハリントンら、Ｃｅｌｌ．Ｉｍｍｕｎｏｌ．、３０：２６１（１９７７））、リウマチ性多発関節炎患者の滑液にも（オディンクら、Ｎａｔｕｒｅ、３３０：８０（１９８７））それから種々の慢性炎症部位にも見出されている（ブルマイスターら、Ｌｙｍｐｈｏｋｉｎｅ Ｒｅｓ．、３：２３６（１９８４））。炎症部位にマクロファージ遊走阻止因子が発現していることは、宿主の防御におけるマクロファージの機能を調節するメディエーターとして、マクロファージ遊走阻止因子が寄与していることが示唆されている。
【０００５】
ヒトマクロファージ遊走阻止因子は、１９８９年にクローニングされた（ワイザーら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．、８６：７５２２（１９８９））。次いで、マウスマクロファージ遊走阻止因子もクローニングされた（ベルンハーゲンら、Ｎａｔｕｒｅ、３６５：７５６（１９９３））。
【０００６】
この後、マクロファージ遊走阻止因子の種々の性質が明らかになってきている。例えば、マクロファージ遊走阻止因子は、脳下垂体前葉の副腎皮質刺激ホルモン産生細胞内の分泌顆粒の主要成分である。また、マクロファージ遊走阻止因子は、エンドトキシンショックに至る宿主反応において中心的な役割を演じているように考えられている（ベルンハーゲンら、Ｎａｔｕｒｅ、３６５：７５６（１９９３））。また、マクロファージ遊走阻止因子は、免疫グロブリンE結合因子の翻訳後のグリコシル化阻害活性を有しており、「グリコシル化阻害因子」とされている（ミカヤマら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．、９０：１００５６（１９９３））。さらに、マクロファージ遊走阻止因子ｍＲＮＡは、胎児の目のレンズや、増殖因子によって活性化された繊維芽細胞にその発現が認められている（ウイストーら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．、９０：１２７２（１９９３）；ラナハンら、Ｍｏｌ．Ｃｅｌｌ．Ｂｉｏｌ．、１２：３９１９（１９９２））。また、マクロファージ遊走阻止因子は、ヒトメラノーマ細胞に発現しており、その腫瘍細胞増殖と血管形成に関与している（シミズら、Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍ．、２６４：７５１（１９９９））。しかし、マクロファージ遊走阻止因子の幹細胞に対する生理作用については、報告された例がない。
【０００７】
【発明が解決しようとする課題】
本発明の目的は、ヒト造血幹細胞および造血前駆細胞を体外で従来よりも効率的に増殖させ得る造血幹細胞増殖剤を提供することにある。本発明のさらなる目的は、各種の造血器官疾患、癌の放射線治療および化学療法の際の造血不全に対する治療薬として利用され得、血球の大量生産に利用され得、遺伝子治療時の造血幹細胞への遺伝子の導入効率の向上に利用され得、さらに診断薬および検査薬として活用され得る、造血幹細胞増殖剤を提供することにある。
【０００８】
【課題を解決するための手段】
本発明者らは、上記目的を達成すべく鋭意検討を重ねた結果、マウス骨髄腫細胞（ＮＳ−１細胞）培養上清に造血幹細胞の増殖を活性化する蛋白質が存在することを見出した。本発明者らは、この蛋白質を分離、精製、および同定することにより、この蛋白質が造血幹細胞増殖因子として新規である、マクロファージ遊走阻止因子であることを明らかにし、それらの造血幹細胞増殖活性を利用することにより、本発明を完成するに至った。
【０００９】
本発明の造血幹細胞増殖剤は、マクロファージ遊走阻止因子を含有する。１つの実施態様では、上記マクロファージ遊走阻止因子は、配列番号１で表わされるアミノ酸配列を有するポリペプチド、配列番号１で表わされるアミノ酸配列において１もしくは数個のアミノ酸が置換、欠失もしくは付加されたアミノ酸配列を有し、かつ造血幹細胞増殖活性を有するポリペプチド、またはこれらの修飾体であり得る。
【００１０】
１つの実施態様では、上記マクロファージ遊走阻止因子は、配列番号２で表わされるアミノ酸配列を有するポリペプチド、配列番号２で表わされるアミノ酸配列において１もしくは数個のアミノ酸が置換、欠失もしくは付加されたアミノ酸配列を有し、かつ造血幹細胞増殖活性を有するポリペプチド、またはこれらの修飾体であり得る。
【００１１】
本発明の造血幹細胞または造血前駆細胞の増殖方法は、上記のいずれかの造血幹細胞増殖剤を用いて非ヒト動物またはインビトロにおいて造血幹細胞または造血前駆細胞を増殖させる工程を包含する。
【００１２】
１つの実施態様では、上記増殖工程において、造血幹細胞または造血前駆細胞に外来遺伝子が導入され得る。
【００１３】
本発明のヒト組織幹細胞または組織前駆細胞の増殖方法は、上記のいずれかの造血幹細胞増殖剤を用いて非ヒト動物またはインビトロにおいてヒト組織幹細胞または組織前駆細胞を増殖させる工程を包含する。
【００１４】
本発明の血液系の疾患の診断薬または検査薬は、上記のいずれかの造血幹細胞増殖剤を含む。
【００１５】
【発明の実施の形態】
本発明の実施においては、特に指示のない限り、当該分野で既知であるタンパク質の分離および分析法、およびアッセイ法が採用される。
Ｉ．定義
以下に、本発明を説明する上で用いられる用語を説明する。マクロファージ遊走阻止因子は、上述のように、テンジクネズミのマクロファージの遊走を阻害するタンパク質として、最初に見出されたリンフォカインである。本発明において、用語「マクロファージ遊走阻止因子」は、いわゆるマクロファージ遊走阻止因子のポリペプチドに限定されず、このポリペプチドに対してアミノ酸配列における実質的な相同性を有するポリペプチド、およびこれらのポリペプチドのいずれかの修飾体もまた含んでいう。相同なポリペプチドの例として、種変異体、および対立遺伝子変異体がある。
【００１６】
ヒト由来のマクロファージ遊走阻止因子は、配列表の配列番号１のアミノ酸配列を含む。マウス由来のマクロファージ遊走阻止因子の場合、配列表の配列番号２のアミノ酸配列を含む。
【００１７】
ヒトの疾患または治療の目的において、およびヒトの造血幹細胞の増殖においてヒト由来のポリペプチドが好ましいことは明らかである。しかし、他の哺乳動物由来の相同なポリペプチドもまた目的に応じて使用可能である。さらに、他の哺乳動物由来のポリペプチドとの比較は、ヒト由来のポリペプチドの所望の活性が保持された改変体を得る上で重要である。
【００１８】
本発明に用いられるマクロファージ遊走阻止因子は、上記の特定の配列によって必ずしも限定されることはなく、これらの配列に対して、１もしくは数個のアミノ酸が欠失、置換もしくは付加されたアミノ酸配列を有し、かつ所望の活性が保持された相同なポリペプチド（すなわち、マクロファージ遊走阻止因子の相同体）も対象として含まれる。ここで、「数個」までのアミノ酸の変異とは、所望の活性が得られる限り、必ずしも特定の上限値に束縛されることを意図しないが、代表的には約６０個以下、好ましくは約４０個以下、より好ましくは約２０個以下の範囲であり得る。アミノ酸の付加の例には、アミノ末端の１個のＭｅｔ残基の付加が含まれる。
【００１９】
アミノ酸の保存的置換は、相同なポリペプチドを得るための好ましい手段の１つである。保存的置換は、代表的には以下のグループ内での置換を包含する：グリシン、アラニン；バリン、イソロイシン、ロイシン；アスパラギン酸、グルタミン酸；アスパラギン、グルタミン；セリン、トレオニン；リジン、アルギニン；およびフェニルアラニン、チロシン。
【００２０】
２つのアミノ酸配列の間の配列同一性（相同性）は、必要であればギャップを導入して、残基の適合を最適化することにより決定される。ヒトのマクロファージ遊走阻止因子に実質的なアミノ酸配列相同性を有するポリペプチドは、ヒトのマクロファージ遊走阻止因子のアミノ酸配列と比較して、代表的には少なくとも約６０％、好ましくは少なくとも約７０％、より好ましくは少なくとも約８０％、さらに好ましくは少なくとも約９０％以上の相同性を有するポリペプチドとして表され得る。相同性決定のためのソフトウェアは、容易に入手可能であり、例えば、Ｇｅｎｅ Ｗｏｒｋｓ（Ｉｎｔｅｌｌｉｇｅｎｅｔｉｃｓ Ｉｎｃ．）などが入手可能である。
【００２１】
本発明に用いられるマクロファージ遊走阻止因子の修飾体としては、上記の配列と同一または相同な配列を有するポリペプチドであって、そのアミノ酸側鎖またはアミノ末端またはカルボキシル末端が修飾され、かつ所望の活性が保持されたポリペプチドが含まれる。マクロファージ遊走阻止因子の相同体と修飾体とを総称して、機能的等価体ともいう。上記の修飾体の例として、アミノ末端がアシル化（例えば、アセチル化）されたポリペプチド、およびカルボキシ末端がアミド化またはエステル化されたポリペプチドが挙げられる。
【００２２】
本発明において「造血幹細胞増殖活性を有する」とは、定義上、下記の実施例１６に実質的に同一の条件（マクロファージ遊走阻止因子の添加濃度は２０ｎｇ／ｍｌとする）で測定したとき、ＣＤ３４抗原が陽性でかつＣＤ３８抗原が陰性の（以下ＣＤ３４＋ＣＤ３８−と表わす）細胞数が実施例１６の対照区に示された細胞数よりも多いこと、代表的には約１１０％以上、好ましくは約２００％以上であることをいう。
【００２３】
「造血幹細胞」とは、赤血球、白血球、巨核球などの骨髄細胞のみならず、T細胞、B細胞などのリンパ系を含めた全ての血液系への分化能を有する多能性の細胞であって、自己増殖可能な細胞をいう。造血幹細胞は、ＣＤ３４抗原が陽性でかつＣＤ３８抗原が陰性である（ＣＤ３４＋ＣＤ３８−）ことにより特徴付けられる。さらにより未熟な造血幹細胞は、ＣＤ３４抗原が陽性でかつＣＤ３８抗原、ＣＤ３３抗原、ＣＤ１９抗原、およびＣＤ２抗原が陰性である（ＣＤ３４＋ＣＤ３８−３３−１９−２−）ことにより特徴付けられる。「造血前駆細胞」とは、血液系の特定の細胞系列への分化が決定付けられているが、分裂により自己増殖可能な細胞をいう。造血前駆細胞は、ＣＤ３４抗原とＣＤ３８抗原とがいずれも陽性である（ＣＤ３４＋ＣＤ３８＋）ことにより特徴付けられる。
【００２４】
「組織幹細胞」とは、分裂に伴って自己再生能を示すとともに、同時に性質の異なる細胞を生じる分化能を有する細胞をいい、造血幹細胞、表皮基底膜の幹細胞、小腸粘膜上皮の幹細胞、神経系幹細胞など、あらゆる種類の幹細胞を含む。「組織前駆細胞」とは、特定の組織に分化および成熟する前段階の細胞をいう。
ＩＩ．造血幹細胞増殖活性を有するマクロファージ遊走阻止因子
本発明においては、マクロファージ遊走阻止因子は、造血幹細胞増殖剤の有効成分として利用される。マクロファージ遊走阻止因子は、天然の供給源から単離されたもの、組換えＤＮＡ技術を使用して産生したもの、または化学合成したものであり得る。
【００２５】
マクロファージ遊走阻止因子を天然の供給源から単離する場合、例えば、以下のようにして精製し得る。まず、骨髄腫細胞（例えば、Ｐ３−ＮＳＩ／１−Ａｇ４−１（略称：ＮＳ−１）細胞）を培養し、得られる培養上清から造血幹細胞増殖活性タンパク質を分離および精製する。「骨髄腫細胞」とは、永久増殖能を有する、癌化した株化細胞である。骨髄腫細胞は、種々の用途が知られており、例えば、ケーラーおよびミルシュタインは、マウス由来の骨髄腫細胞であるＰ３−Ｘ６３−Ａｇ８（略称Ｘ６３）細胞を、ヒツジ赤血球細胞で感作したマウス脾臓由来のリンパ球と細胞融合し、B細胞ハイブリドーマを作製した（ケーラーら、Ｎａｔｕｒｅ、２５６：４９５（１９７５））。Ｘ６３細胞は、ＢＡＬＢ／ｃマウス由来の免疫グロブリンＧ１カッパ鎖産生骨髄腫細胞から、８−アザグアニン耐性であり、ヒポキサンチン、アミノプテリンおよびチミジンを含む培地では増殖できない細胞集団を、クローニングにより選択して得られた細胞株である。ＮＳ−１細胞は、Ｘ６３細胞由来の骨髄腫細胞で、免疫グロブリンＧ１カッパ鎖のみを合成するが、分泌はしないことが知られている（ケーラーら、Ｅｕｒ．Ｊ．Ｉｍｍｕｎｏｌｏｇｙ、６：５１１（１９７６））。
【００２６】
本発明者らは、このＮＳ−１細胞培養上清に、驚くべきことに、造血幹細胞増殖活性が存在することを見出した。従って、本発明における造血幹細胞増殖活性因子は、ＮＳ−１細胞の培養上清、または同様な活性を示す他の骨髄腫細胞の培養上清から単離することができる。骨髄腫細胞培養上清に見出された造血幹細胞増殖活性タンパク質の分離精製は、造血幹細胞増殖活性を示す骨髄腫細胞培養上清を大量に調製することから始める。細胞の培養は、通常の株化細胞の培養方法に準じればよい。例えば、適当な比率の牛胎児血清（ＦＣＳ）を含有する動物細胞培養用培地中で、３７℃に設定したＣＯ2インキュベーターで培養する。
【００２７】
培養上清からタンパク質を分離および精製するためには、夾雑タンパク質をできるだけ減少させる必要がある。そのために、最も造血幹細胞増殖活性タンパク質の含有量が増える直前期に、培地を無血清培地に交換して、培養を継続し、その後培養上清を回収することが好ましい。回収した培養上清をフィルターに通して、細胞残渣を除くことができる。
【００２８】
得られた培養上清から、造血幹細胞増殖活性タンパク質を分離および精製するためには、通常のタンパク質の分離精製方法を用いることができる。培養上清を濃縮するためには、限外ろ過膜を用い得る。濃縮した培養上清を、ＨＰＬＣを用いたゲルろ過カラムにアプライし、造血幹細胞増殖活性を有する画分を回収する。得られた活性画分を逆相ＨＰＬＣカラムに供する。溶出したものの中から、造血幹細胞増殖活性を有する画分を回収する。こうして得られた画分は、充分な純度を有し、さらなる精製を行わずにこのままプロテインシークエンサーによるアミノ酸配列分析に供することができる。こうして、骨髄腫細胞培養上清に見出される造血幹細胞増殖活性タンパク質を同定することができる。
ＩＩＩ．造血幹細胞増殖活性の確認
次にマクロファージ遊走阻止因子の造血幹細胞増殖活性の確認の方法の例を説明する。臍帯血から調製した造血幹細胞（ＣＤ３４＋ＣＤ３８−３３−１９−２−細胞）を、マイクロタイタープレートにプレーティングする。造血幹細胞の増殖に使用する培地としては、試験区では、１５％ＦＣＳ−ＲＰＭＩ１６４０に、サイトカインである幹細胞成長因子、インターロイキン６、およびインターロイキン３を添加した培地（幹細胞成長因子／インターロイキン６／インターロイキン３培地）を用意し、これに造血幹細胞増殖活性を検定しようとする目的のポリペプチド（例えばヒトマクロファージ遊走阻止因子）を含有するサンプルを添加した培地を用いる。対照区では、目的のポリペプチドを含有する上記サンプルの代わりに、２％ＦＣＳ−ＲＰＭＩ１６４０を幹細胞成長因子／インターロイキン３培地に加えた培地を用いる。これらを約３７℃で１０〜３０日間程度培養した後、細胞にＣＤ３４−ＦＩＴＣ抗体（ＣＤ３４抗体にフルオレシンイソチアシネート色素を結合したもの）処理を行い、各一定時間ＦＡＣＳｃａｎにて細胞の取り込みを行いＣＤ３４＋細胞比率を求め、生細胞をトリパンブルー法で測定し、生細胞数にＣＤ３４＋細胞比率を乗じて算出することにより、ＣＤ３４＋細胞数を求める。このようにして、目的ポリペプチドの造血幹細胞増殖活性の確認ができる。
ＩＶ．造血幹細胞増殖剤およびその利用
本発明の造血幹細胞増殖剤は、造血幹細胞増殖活性を有するマクロファージ遊走阻止因子と、その活性を実質的に阻害しない任意の媒体とを含有する組成物として提供される。ここで「マクロファージ遊走阻止因子」は広義に用いられ、通常のマクロファージ遊走阻止因子の他、上述したマクロファージ遊走阻止因子の相同体および修飾体を含む意である。ヒトへの投与のための造血幹細胞増殖剤は、代表的には、有効量のマクロファージ遊走阻止因子に加えて、当業者に公知の任意の薬学的に受容可能な賦形剤を含有し得る。賦形剤の例としては、乳糖、コーンスターチ、ステアリン酸マグネシウム、ミョウバンなどが挙げられる。
【００２９】
本発明の造血幹細胞増殖剤は、当該分野で公知の方法に従って調製され得る。本発明の造血幹細胞増殖剤は、任意の形状であり得る。本発明の造血幹細胞増殖剤は、錠剤、丸剤、カプセル剤、顆粒剤のような固体；または水溶液および懸濁液のような液体であり得る。本発明の造血幹細胞増殖剤を錠剤として経口投与する場合、通常、乳糖、コーンスターチ、およびステアリン酸マグネシウムのような賦形剤が使用され得る。本発明の造血幹細胞増殖剤をカプセル剤として経口投与する場合、通常、乳糖および乾燥コーンスターチのような賦形剤が使用され得る。水性懸濁液として経口投与するためには、マクロファージ遊走阻止因子を乳濁液または懸濁液と組み合わせて使用し得る。水性懸濁液は、必要に応じて、甘味剤および香料を含有し得る。本発明の造血幹細胞増殖剤を筋肉内、腹腔内、皮下、および静脈内注射する場合は、滅菌した溶液にマクロファージ遊走阻止因子を溶解させて緩衝液を調製し、ｐＨを適切な値に調節する。本発明の造血幹細胞増殖剤を静脈内投与する場合は、この増殖剤は等張であることが好ましい。
【００３０】
本発明の造血幹細胞増殖剤を用いることにより、造血幹細胞または造血前駆細胞を含む組織幹細胞または組織前駆細胞を大量に増殖させることが可能である。例えば、本発明の造血幹細胞増殖剤を用いて、インビトロにおいて造血幹細胞または造血前駆細胞を増殖させることにより、血球を大量生産することができる。造血幹細胞増殖剤の有効量ならびに血球の生産および回収の条件などは、当業者により適宜選択され得る。
【００３１】
さらに、本発明の造血幹細胞増殖剤は、免疫抑制障害などの各種の造血器官疾患、ならびに癌の放射線治療および化学療法による造血不全のような、血球の増加が所望される患者の治療に用いることができる。治療用途において、本発明の造血幹細胞増殖剤は、Ｒｅｍｉｎｇｔｏｎ‘ｓ ＰｈａｒｍａｃｅｕｔｉｃａｌＳｃｉｅｎｃｅｓ、 Ｍａｃｋ Ｐｕｂｌｉｓｈｉｎｇ社（Ｅａｓｔｏｎ、ＰＡ）に記載されているような従来のポリペプチドの処方物の形で投与され得る。例えば、本発明の造血幹細胞増殖剤は、経口投与、静脈投与、筋肉注射、腹腔内注射、および皮下注射のような非経口投与により投与され得る。これらのポリペプチドを羊水中へ補充することも可能である。好ましくは、これらのポリペプチドは、注射によって投与され得る。
【００３２】
本発明の造血幹細胞増殖剤を、ヒトに投与する場合、１日あたりの用量は、通常、患者の症状、重篤度、感受性に対する個体差、体重、年齢などを考慮して、当業者によって適切に決定され得る。本発明の造血幹細胞増殖剤は、１日１回投与されてもよいし、１日数回に分けて投与されてもよい。
【００３３】
遺伝子治療においては、目的の遺伝子の導入効率は、遺伝子を細胞に導入する工程で、どれだけ多数の細胞が分裂を行っているかに左右されることが知られている。従って、本発明の造血幹細胞増殖剤を用いて造血幹細胞または造血前駆細胞を増殖させつつ遺伝子を導入することにより、非常に高い遺伝子導入効率を得ることができる。
【００３４】
さらに、本発明の造血幹細胞増殖剤は、そのままで血液系の疾患（例えば、白血病、骨髄腫、貧血など）の診断薬および検査薬として用いることができる。
【００３５】
【実施例】
以下、本発明における造血幹細胞増殖剤としてのマクロファージ遊走阻止因子についてさらに具体的に説明する。
【００３６】
（実施例１：ヒト臍帯血からの、造血幹細胞の調製）
産婦人科からインフォームドコンセントにて供与された、ヒト臍帯血（抗凝固剤としてヘパリンを用いたもの約５０ｍｌ）からフィコール法（アマシャムファルマシアバイオテク社製、フィコールパックを用いた）にて、単核球画分を調製し、凍結保存した。これをアッセイ開始時に凍結融解後、１５％ＦＣＳ−ＲＰＭＩ１６４０（株式会社日研生物医学研究所製）にて洗浄し、細胞に、ＣＤ３４−ＦＩＴＣ抗体（ＣＤ３４抗体にフルオレシンイソチアシネート色素を結合したもの）、ＣＤ３８−ＰＥ抗体（ＣＤ３８抗体にフィコエリスリン色素を結合したもの）、ＣＤ３３−ＰＥ抗体（ＣＤ３３抗体にフィコエリスリン色素を結合したもの）、ＣＤ１９−ＰＥ抗体（ＣＤ１９抗体にフィコエリスリン色素を結合したもの）、およびＣＤ２−ＰＥ抗体（ＣＤ２抗体にフィコエリスリン色素を結合したもの）の各抗体（イムノテック社製）処理を行い、ＦＡＣＳｔａｒ（ベクトンディッキンソン社製）にて、造血幹細胞ＣＤ３４＋ＣＤ３８−３３−１９−２−細胞を分取した。
【００３７】
（実施例２：ヒト臍帯血の造血幹細胞を用いた造血幹細胞増殖因子検出系の構築）
実施例１で分取したヒト臍帯血のＣＤ３４＋ＣＤ３８−３３−１９−２−細胞を９６ウエルＵ型プレート（コースター社製）にプレーティングした（５０細胞／１００μｌ／ウエル）。用いた培地は、１５％ＦＣＳ−ＲＰＭＩ１６４０に、サイトカインである幹細胞成長因子（２５ｎｇ／ｍｌ）、インターロイキン６（２０ｎｇ／ｍｌ）、およびインターロイキン３（２０ｎｇ／ｍｌ）を添加したものであり、さらに造血幹細胞増殖活性について測定しようとするサンプル２０μｌ（／ウエル）を添加した。
３７℃で１４〜２７日液体培養後、生細胞数をトリパンブルー法（トリパンブルーは大日本製薬社製）にて測定した。細胞のカウントは血球計算盤を用いた。
ＣＤ３４＋細胞数およびＣＤ３４＋ＣＤ３８−細胞数は、以下の通りに算出した。まず、液体培養後の細胞をＣＤ３４−ＦＩＴＣ抗体（イムノテック社製）およびＣＤ３８−ＰＥ抗体（イムノテック社製）（細胞１０5個に対して抗体５０ｎｇ）で染色した。染色された細胞を、ＰＢＳ（−）（ｐｈｏｓｐｈａｔｅ−ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ、ｐＨ７．２；株式会社日研生物医学研究所製）（ＦＣＳ１％、アジ化ナトリウム０．０１％を含む）で２回洗浄した後、ヨウ化プロピジウム（シグマアルドリッチジャパン株式会社製）を最終濃度１０μｇ／ｍｌになるように加えて細胞を染色した。染色された細胞をフローサイトメーターＦＡＣＳｃａｎ（ベクトンディッキンソン社製）で解析（フローサイトメトリー）して、ＣＤ３４＋細胞比率およびＣＤ３４＋ＣＤ３８−細胞比率を求め、生細胞数にその比率を乗じることにより、ＣＤ３４＋細胞数およびＣＤ３４＋ＣＤ３８−細胞数を算出した。
【００３８】
（実施例３：マウスミエローマ細胞培養およびその培養上清の調製）
マウスミエローマ細胞（ＮＳ−１）培養は、細胞１．０×１０5／ｍｌの濃度になるように、１５％ＦＣＳ−ＲＰＭＩ１６４０培地１５ｍｌに懸濁し、カルチャーディッシュ（グライナー社製）（直径１０ｃｍ）に入れ、５％ＣＯ2インキュベーターで、３７℃で培養を開始した。培養０、１、２、３、４、５、６、７および８日目に培養上清を回収し、０．２２μｍのフィルターを通過した上清を集めた。
【００３９】
（実施例４：ＮＳ−１細胞増殖曲線の作製）
ＮＳ−１細胞培養０、１、２、３、４、５、６、７および８日目に、生細胞数および死細胞数をトリパンブルー法にて測定した。測定の結果、生細胞濃度は、それぞれ１×１０5個／ｍｌ、４×１０5個／ｍｌ、１０×１０5個／ｍｌ、１７×１０5個／ｍｌ、１８×１０5個／ｍｌ、１４×１０5個／ｍｌ、１０×１０5個／ｍｌ、５×１０5個／ｍｌ、２×１０5個／ｍｌであった。死細胞濃度は、０．０５×１０5個／ｍｌ、０．１×１０5個／ｍｌ、０．５×１０5個／ｍｌ、１×１０5個／ｍｌ、４×１０5個／ｍｌ、１０×１０5個／ｍｌ、１３×１０5個／ｍｌ、１７×１０5個／ｍｌ、２０×１０5個／ｍｌであった。この結果、培養4日目に生細胞数が最大になることがわかった。
【００４０】
（実施例５：ＮＳ−１細胞培養上清回収時期の決定）
ＮＳ−１細胞を培養して、培養０、１、２、３、４、５、６、７および８日目に回収した培養上清を、実施例２で構築したヒト臍帯血造血幹細胞を用いた造血幹細胞増殖因子検出系を用いて評価を行った。各培養上清を９６穴Ｕ型ウエルに２０μｌずつ添加した。ＣＤ３４＋細胞数は、培養４、５、６、７そして８日目ではそれぞれ、３６２個、５５０個、１５３個、１１５個および９３個であった。その結果、培養５日目の上清に最も造血幹細胞増殖活性が存在することがわかった。
【００４１】
（実施例６：ＮＳ−１細胞培養上清の造血幹細胞増殖活性の濃度依存性についての検討）
実施例３で回収したＮＳ−１細胞培養上清の造血幹細胞増殖活性の濃度依存性について検討した。ヒト臍帯血造血幹細胞を用いた造血幹細胞増殖因子検出系を用いて、培養２５日目のＮＳ−１細胞培養上清を、原液のまま、２倍希釈、４倍希釈、８倍希釈、１６倍希釈、３２倍希釈、６４倍希釈、１２８倍希釈および２５６倍希釈して添加して造血幹細胞増殖活性を評価した。ＣＤ３４＋細胞数はそれぞれ、２３８個、１４４個、７４個、１０４個、４５個、１個、０個、１１個および１０個であった。ＣＤ３４＋ＣＤ３８−細胞数はそれぞれ、１０７個、３６個、４４個、３７個、１７個、１個、０個、２個および１個であった。このように造血幹細胞の増殖はＮＳ−１細胞培養上清の濃度にほぼ依存していた。
【００４２】
（実施例７：ＮＳ−１細胞培養上清の大量調製）
ＮＳ−１細胞を１．０×１０5／ｍｌの濃度になるように、１５％ＦＣＳ−ＲＰＭＩ１６４０培地５０ｍｌに懸濁し、カルチャーフラスコ（グライナー社製、１５０ｃｍ2）に入れた。５％ＣＯ2インキュベーターで、３７℃の条件で培養を開始し、培養後５日目の培養上清を回収し、０．２２μｍのフィルター（日本ミリポア社製）を通過した上清を集めた。合計３，６２０ｍｌの培養上清を回収した。
【００４３】
（実施例８：ＮＳ−１細胞培養上清のゲルろ過による精製）
実施例７で回収したＮＳ−１細胞培養上清１５ｍｌを、分画分子量１０，０００の限外ろ過フィルター（ゲルマンサイエンシズ社製）で１ｍｌに濃縮後、全量をＳｅｐｈａｃｒｙｌ Ｓ−２００カラム（アマシャムファルマシアバイオテク社製）（直径１０ｍｍ、高さ１９．１ｃｍ）にアプライした。平衡化緩衝液には、ＲＰＭＩ１６４０培地（日水製薬製）を用いて、溶出画分を集めた。
溶出画分を、実施例２で構築したヒト臍帯血造血幹細胞を用いた造血幹細胞増殖因子検出系で造血幹細胞増殖活性について評価をしたところ、分子量２万付近の画分に活性が認められた。
５日間３７℃で保存した１５％ＦＣＳ−ＲＰＭＩ１６４０培地を同様にしてＳｅｐｈａｃｒｙｌ Ｓ−２００カラムでゲルろ過したものを対照にして、同じ造血幹細胞増殖因子検出系で評価したが、対照では造血幹細胞増殖活性はほとんど認められなかった。
以上から、分子量１〜２万付近の培養上清画分に造血幹細胞増殖活性因子が存在することがわかった。
【００４４】
（実施例９：ＮＳ−１細胞培養上清のゲルろ過画分の逆相ＨＰＬＣによる精製）
実施例８で得られたゲルろ過での活性画分を逆相ＨＰＬＣに供した。逆相ＨＰＬＣカラムとして、ＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰ（株式会社ワイエムシィ製、長さ２５０ｍｍ、内径４．６ｍｍ）を用い、ＨＰＬＣ装置は島津製作所製ＬＣ−１０Ａを使った。溶出はトリフルオロ酢酸（ＴＦＡ）−アセトニトリル（ＣＨ3ＣＮ）の系で行った。すなわちＡ液には０．１％ＴＦＡ（ｐＨ２．０）、Ｂ液には８０％ＣＨ3ＣＮ−０．１％ＴＦＡ（ｐＨ２．０）を用いて、最初の５分間はＢ液の濃度を０％、次の７０分間でＢ液の濃度を０％から７０％に直線グラジエントで、次の１０分間で７０％から１００％まで同様に直線グラジエントで溶出した。溶出画分を、ヒト臍帯血造血幹細胞を用いた造血幹細胞増殖因子検出系で評価を行ったところ、リテンション時間６０分付近の画分および６３分付近の画分に活性が認められた。
【００４５】
（実施例１０：無血清培養によるＮＳ−１細胞培養およびその培養上清の調製）
１５％ＦＣＳ含有培地よりも、より精製が容易な培養上清を作製するために、無血清培養によるＮＳ−１細胞培養方法を確立した。ＮＳ−１細胞を１×１０5／ｍｌの濃度で１５％ＦＣＳ−ＲＰＭＩ１６４０の培地４０ｍｌで、直径１４．５ｃｍのディシュ（グライナー社製）にて培養開始した。培養４日目に新しい１５％ＦＣＳ−ＲＰＭＩ１６４０の培地４０ｍｌに交換し、さらに１日培養した。次いで、ＰＢＳ（−）で２回洗浄し、血清を含まないＲＰＭＩ１６４０の培地４０ｍｌに移し、新しいディッシュで１日間培養した。次いで、培養物を０．２２μｍのフィルターを通し、細胞残渣を除去して、培養上清を回収した。回収した培養上清を、ヒト臍帯血造血幹細胞を用いた造血幹細胞増殖因子検出系での評価に供して活性が存在することを確認した。
【００４６】
（実施例１１：ＮＳ−１細胞無血清培養上清のゲルろ過による精製）
実施例１０で回収したＮＳ−１細胞無血清培養上清２０ｍｌを分画分子量１０，０００の限外ろ過フィルター（ゲルマンサイエンシズ社製）で１／１０に濃縮して得た濃縮培養上清２ｍｌを、ＴＳＫｇｅｌ Ｇ３０００ＳＷXLカラムにアプライした。平衡化緩衝液には、０．３Ｍ食塩を含む５０ｍＭリン酸緩衝液を用い、溶出画分を集めた。溶出画分を、ヒト臍帯血造血幹細胞を用いた造血幹細胞増殖因子検出系で評価を行ったところ、分子量１〜２万付近の画分に活性が認められた。
【００４７】
（実施例１２：ＮＳ−１細胞無血清培養上清のゲルろ過画分の逆相ＨＰＬＣによる精製）
実施例１１で得られたゲルろ過での活性画分１４ｍｌを逆相ＨＰＬＣに供した。逆相ＨＰＬＣカラムとして、ＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰ（株式会社ワイエムシィ製、長さ２５０ｍｍ、内径４．６ｍｍ）を用い、ＨＰＬＣ装置は島津製作所製ＬＣ−１０Ａを使った。溶出はＴＦＡ−ＣＨ3ＣＮの系で行った。すなわちＡ液には０．１％ＴＦＡ（ｐＨ２．０）、Ｂ液には８０％ＣＨ3ＣＮ−０．１％ＴＦＡ（ｐＨ２．０）を用いて、最初の１５分間はＢ液の濃度を０％、次の７０分間でＢ液の濃度を０％から７０％に直線グラジエントで、次の１０分間で７０％から１００％まで同様に直線グラジエントで溶出した。
ＣＨ3ＣＮ濃度が４７％付近のピーク（リテンション時間が７１分、７２分、および７４分の合計３つ）を回収して、再度同じカラムでリクロマトグラフィーを行い同様に精製した。
【００４８】
（実施例１３：精製サンプルのアミノ末端アミノ酸配列確認）
上記の実施例１２においてＹＭＣ−Ｐａｃｋ ＰＲＯＴＥＩＮ−ＲＰのリクロマトグラフィーで精製して得られた３つのサンプルを、プロテインシークエンサー（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ Ｐｒｏｃｉｓｅ Ｓｅｑｕｅｎｃｅｒ：Ｍｏｄｅｌ ４９２）にアプライして、分析した。
７１分のものは、２５アミノ酸残基のアミノ末端分析で、マウスサイクロフィリンＢに一致した。７２分のものは、マウスサイクロフィリンＡに一致した。７４分のものは、その主たる構成タンパク質はエドマン分解できない構造と思われた。しかし、マイナーな構成タンパク質としては、マウスマクロファージ遊走阻止因子に一致した。リテンション時間が７４分のサンプルについて、トシルフェニルアラニルクロロメチルケトン（ＴＰＣＫ）−トリプシンを用いたペプチドマップを作製して分析したところ、アミノ末端のペプチドがエドマン分解できなかったが、その他のトリプシンペプチドはマウスサイクロフィリンＡに一致した。このタンパク質はマウスサイクロフィリンＡ様タンパク質と考えられた。
【００４９】
（実施例１４：リテンション時間７４分のサンプル中の混合物の分離）
実施例１３で、リテンション時間が７４分のサンプル中で、マイナーな構成タンパク質であるマウスマクロファージ遊走阻止因子と、主たる構成タンパク質であるマウスサイクロフィリンＡ様タンパク質が混合していることがわかったので、両者の分離を試みた。実施例１０、１１および１２の方法で数ロットのＮＳ−１細胞培養上清の精製を行い、逆相ＨＰＬＣのクロマトパターンを調べたところ、いくつかのロットにおいて、リテンション時間が７４分のピークに相当するピークが２つに分かれることがわかった。そのうちリテンション時間が遅いピークをプロテインシークエンサーで解析したところ、このピークはマウスマクロファージ遊走阻止因子であることがわかった（図１）。また、同様にＴＰＣＫ−トリプシンを用いたペプチドマッピングとプロテインシークエンサーでの解析で、リテンション時間が早いピークの方は、マウスサイクロフィリンＡ様タンパク質であることもわかった。
【００５０】
（実施例１５：精製サンプルの造血幹細胞増殖活性確認）
未熟な造血幹細胞であるＣＤ３４＋ＣＤ３８−細胞数の増幅を指標に、実施例１４で得たマウスマクロファージ遊走阻止因子画分の活性を、実施例２で構築した造血幹細胞増殖因子検出系を用いて調べた。対照としては、２％ＦＣＳ−ＲＰＭＩ１６４０を用いた。それぞれ、９６穴ウエルの５ウエルで培養した細胞を混合して、この中に含まれる生細胞数を、実施例２で説明したトリパンブルー法で測定した。ＣＤ３４＋細胞数およびＣＤ３４＋ＣＤ３８−細胞数の算出についても、実施例２で説明した方法で算出した。マウスマクロファージ遊走阻止因子画分の濃度は正確には決定されなかったが、２〜２０ｎｇ／ｍｌ程度の濃度であった。
結果は、全体の生細胞数、ＣＤ３４＋細胞数、およびＣＤ３４＋ＣＤ３８−細胞数ともに対照区に比べて増幅していた（代表的な結果を図２および図３に示す）。全体の生細胞数で、対照区に比べて１８．８倍、ＣＤ３４＋細胞数で対照区に比べて３．８倍、そしてＣＤ３４＋ＣＤ３８−細胞数で対照区に比べて３．４倍に増幅していた。このことは、マウスマクロファージ遊走阻止因子に、ヒト臍帯血造血幹細胞増殖活性が存在することを示している。
【００５１】
（実施例１６：大腸菌型ヒトマクロファージ遊走阻止因子の造血幹細胞増殖活性の確認）
ヒト臍帯血から調製した造血幹細胞（ＣＤ３４＋ＣＤ３８−細胞）を９６ウエルＵ型プレートに各５ウエルにプレーティング（２００細胞／１００μｌ／ウエル）した。造血幹細胞の増殖に使用した培地は、試験区では、１５％ＦＣＳ−ＲＰＭＩ１６４０に、サイトカインである幹細胞成長因子（２５ｎｇ／ｍｌ）、インターロイキン６（２０ｎｇ／ｍｌ）、およびインターロイキン３（２０ｎｇ／ｍｌ）を添加した培地（幹細胞成長因子／インターロイキン６／インターロイキン３培地）を用意し、これにさらに大腸菌型ヒトマクロファージ遊走阻止因子（Ｒ＆Ｄシステムズ社製）を含有するサンプル（ヒトマクロファージ遊走阻止因子を、２％ＦＣＳ−ＲＰＭＩ１６４０に溶解させたもの）を２０μｌ（／ウエル）添加（ヒトマクロファージ遊走阻止因子の最終濃度は、２０ｎｇ／ｍｌ）した培地であった。対照区では、ヒトマクロファージ遊走阻止因子を含有する上記サンプルの代わりに、幹細胞成長因子／インターロイキン６／インターロイキン３培地に、２％ＦＣＳ−ＲＰＭＩ１６４０を２０μｌ（／ウエル）添加した培地を用いた。本実験はトリプリケイトで実施した。
３７℃で１４日間、および２７日間培養した後、５ウエルを１つにあわせ、この中に含まれる生細胞数を、実施例２で説明したトリパンブルー法で測定した。ＣＤ３４＋細胞数およびＣＤ３４＋ＣＤ３８−細胞数の算出についても、実施例２で説明した方法で算出した。その結果、培養１４日目、および２７日目で明らかに、生細胞数、ＣＤ３４＋細胞数、およびＣＤ３４＋ＣＤ３８−細胞数いずれも、試験区（ヒトマクロファージ遊走阻止因子添加区）は、対照区に比べて、細胞数が増加していた（図４および図５）。図４の場合は、全体の生細胞数で、対照区に比べて２５．３倍、ＣＤ３４＋細胞数で対照区に比べて１３．４倍、そしてＣＤ３４＋ＣＤ３８−細胞数で対照区に比べて１０．５倍に増幅していた。図５の場合は、全体の生細胞数で、対照区に比べて２１．３倍、ＣＤ３４＋細胞数で対照区に比べて１８．０倍、そしてＣＤ３４＋ＣＤ３８−細胞数で対照区に比べて１５．２倍に増幅していた。このことは、大腸菌型ヒトマクロファージ遊走阻止因子に、ヒト臍帯血造血幹細胞増殖活性が存在することを示している。
【００５２】
【発明の効果】
本発明により、マクロファージ遊走阻止因子を含有する造血幹細胞増殖剤が提供される。この造血幹細胞増殖剤は、各種の造血器官疾患、癌の放射線治療および化学療法の際の造血不全に対する治療薬として利用され得、血球の大量生産に利用され得、遺伝子治療時に造血幹細胞への遺伝子の導入効率の向上に利用され得、さらに診断薬および検査薬として活用され得る。
【００５３】
【配列表】
【図面の簡単な説明】
【図１】 ＮＳ−１細胞無血清培養上清のゲルろ過画分の逆相ＨＰＬＣ（ＰＲＯＴＥＩＮ−ＲＰ）による精製クロマトパターンを、その中でマウスマクロファージ遊走阻止因子の画分を明示して示した図である。図１は、縦軸に２１４ｎｍでの吸収を、そして横軸に逆相ＨＰＬＣ（ＰＲＯＴＥＩＮ−ＲＰ）リテンション時間(分)を示す。２１４ｎｍでの吸収は、それぞれの画分に含まれるタンパク質量を反映する。
【図２】 ＮＳ−１細胞無血清培養上清から精製した、マウスマクロファージ遊走阻止因子画分を用いて１５日間培養し、増幅した細胞のＣＤ３４抗原およびＣＤ３８抗原の発現状況を、フローサイトメトリー図により示す。縦軸はＣＤ３８−ＰＥ抗体の蛍光強度、そして横軸はＣＤ３４−ＦＩＴＣ抗体の蛍光強度である。図の左は対照区（２％ＦＣＳ−ＲＰＭＩ１６４０）、右はマクロファージ遊走阻止因子を用いて培養し増幅した細胞のフローサイトメトリー図である。ドットは細胞を示す。十字の線は、上述のようにして培養し増幅した細胞を、マウス免疫グロブリンＧ１−ＦＩＴＣ抗体およびマウス免疫グロブリンＧ１−ＰＥ抗体で処理して得られたフローサイトメトリー図に引いた縦軸と横軸の、それぞれの陽性陰性を区分する線を示す。すなわち右下の象限は、ＣＤ３４＋ＣＤ３８−を示し、右上の象限は、ＣＤ３４＋ＣＤ３８＋を示し、左上の象限は、ＣＤ３４−ＣＤ３８＋を示し、そして左下の象限は、ＣＤ３４−ＣＤ３８−を示す。全体の生細胞数、ＣＤ３４＋細胞数、およびＣＤ３４＋ＣＤ３８−細胞数ともに、対照区に比べて明らかに増幅していることがわかる。
【図３】 ＮＳ−１細胞無血清培養上清から精製したマウスマクロファージ遊走阻止因子画分の、ヒト臍帯血造血幹細胞増殖活性を、まとめた図である。縦軸は細胞数(個)を、横軸は左から順番に１５日目の生細胞、１５日目のＣＤ３４＋細胞、そして１５日目のＣＤ３４＋ＣＤ３８−細胞を示す。黒いカラムは、対照区、白いカラムは、マクロファージ遊走阻止因子を用いて培養したときの細胞数を表す。全体の生細胞数で、対照区に比べて１８．８倍、ＣＤ３４＋細胞数で対照区に比べて３．８倍、そしてＣＤ３４＋ＣＤ３８−細胞数で対照区に比べて３．４倍に増幅しており、マウスマクロファージ遊走阻止因子に、ヒト臍帯血造血幹細胞増殖活性が存在することを示している。
【図４】 大腸菌型ヒトマクロファージ遊走阻止因子の、ヒト臍帯血造血幹細胞増殖活性（培養１４日目）を、まとめた図である。縦軸は細胞数(個)を、横軸は左から順番に１４日目の生細胞、１４日目のＣＤ３４＋細胞、そして１４日目のＣＤ３４＋ＣＤ３８−細胞を示す。縦のバーはそれぞれの標準偏差を示す。黒いカラムは、対照区、白いカラムは、マクロファージ遊走阻止因子を用いて培養したときの細胞数を表す。全体の生細胞数で、対照区に比べて２５．３倍、ＣＤ３４＋細胞数で対照区に比べて１３．４倍、そしてＣＤ３４＋ＣＤ３８−細胞数で対照区に比べて１０．５倍に増幅しており、大腸菌型ヒトマクロファージ遊走阻止因子に、ヒト臍帯血造血幹細胞増殖活性が存在することを示している。
【図５】 大腸菌型ヒトマクロファージ遊走阻止因子の、ヒト臍帯血造血幹細胞増殖活性（培養２７日目）を、まとめた図である。縦軸は細胞数(個)を、横軸は左から順番に２７日目の生細胞、２７日目のＣＤ３４＋細胞、そして２７日目のＣＤ３４＋ＣＤ３８−細胞を示す。縦のバーはそれぞれの標準偏差を示す。黒いカラムは、対照区、白いカラムは、マクロファージ遊走阻止因子を用いて培養したときの細胞数を表す。全体の生細胞数で、対照区に比べて２１．３倍、ＣＤ３４＋細胞数で対照区に比べて１８．０倍、そしてＣＤ３４＋ＣＤ３８−細胞数で対照区に比べて１５．２倍に増幅しており、大腸菌型ヒトマクロファージ遊走阻止因子に、ヒト臍帯血造血幹細胞増殖活性が存在することを示している。[0001]
BACKGROUND OF THE INVENTION
The present invention contains a macrophage migration inhibitory factor as a hematopoietic stem cell growth factor having hematopoietic stem cell survival maintenance activity, proliferative activity, and differentiation activity, and hematopoiesis during various hematopoietic organ diseases, cancer radiotherapy and chemotherapy The present invention relates to a hematopoietic stem cell proliferating agent that can be used as a therapeutic agent for failure. The hematopoietic stem cell proliferating agent of the present invention can also be used for mass production of blood cells and improvement of gene introduction efficiency into hematopoietic stem cells during gene therapy, and further can be used as a diagnostic agent and a test agent.
[0002]
[Prior art]
Hematopoietic stem cells have the ability to differentiate into all mature blood cells (pluripotency) and the ability to replicate cells having the same ability as self (self-renewal ability), and continue to support hematopoiesis for a long time. Hematopoiesis in the human body is generally considered to be regulated by direct interactions between cells and various substances present in body fluids (ie, humoral hematopoietic regulators). Direct interactions between cells include interactions between hematopoietic stem cells, hematopoietic progenitor cells committed to differentiate into specific blood cells, and stromal cells that are the hematopoietic microenvironment surrounding them.
[0003]
Various humoral hematopoietic regulators have been discovered by the progress of biotechnology in the last decades. For example, erythropoietin (Lin et al., Proc. Natl. Acad. Sci. USA, 82: 7580 (1985)), granulocyte colony stimulating factor (Nagata et al., EMBO J, 5: 575 (1986)), Granulocyte-macrophage colony-stimulating factor (Miyatake et al., EMBO J, 4: 2561 (1985)), macrophage colony-stimulating factor (Wong et al., Science, 235: 1504 (1987)), thrombopoietin (Sovage et al., Nature, 369: 533) 1994)), stem cell growth factor (Anderson et al., Cell, 63: 235 (1990)), Fms-like tyrosine kinase 3 ligand (Limanet et al., Cell, 75: 1157 (1993)), leukemia inhibitory factor (Star et al., J. Biol. Biol.Chem., 26 (15): 8833 (1990)), interleukin 1 (Clark et al., Nucleic Acids Res., 14, 7897 (1986)), interleukin 3 (Dorsers et al., Gene, 55: 115 (1987)), inter Leukine 6 (Yaskawa et al., EMBO J, 6: 2939 (1987)), interleukin 11 (Paul et al., Proc. Natl. Acad. Sci. USA, 87: 7512 (1990)), interleukin 12 (Wolf et al., J. Immunol. 146: 3074 (1991)). None of these cytokines can proliferate hematopoietic stem cells by themselves, and in order to proliferate to some extent, it is necessary to use a combination of two or more, preferably three or more. Therefore, it is desired to provide a blood cell growth factor that can proliferate hematopoietic stem cells more efficiently.
[0004]
Macrophage migration inhibitory factor is the first lymphokine found as a protein that inhibits macrophage migration in guinea pigs (Bloom et al., Science, 153: 80 (1966); David et al., Proc. Natl. Acad. Sci. U.S.A., 56:72 (1966)). In addition, expression of macrophage migration inhibitory factor activity is associated with delayed-type hypersensitivity and cellular immunity in animal models and humans (Broom et al., Science, 153: 80 (1966); David et al., Proc. Natl. Acad. Sci.U.S.A., 56:72 (1966); David et al., Prog. Allergy, 16: 300 (1972); Rocklin et al., N. Engl. J. Med., 282: 1340 (1970). ). In addition, macrophage migration inhibitory factor activity is detected in leukocyte culture supernatants upon rejection of mouse xenografts (Arascali et al., Nature, 205: 916 (1965); Harrington et al., Cell. Immunol., 30: 261 (1977)), as well as in the synovial fluid of patients with rheumatoid polyarthritis (Oding et al., Nature, 330: 80 (1987)) and in various sites of chronic inflammation (Blumister et al., Lymphokine). Res., 3: 236 (1984)). The expression of a macrophage migration inhibitory factor at the site of inflammation suggests that the macrophage migration inhibitory factor contributes as a mediator that regulates the function of macrophages in host defense.
[0005]
Human macrophage migration inhibitory factor was cloned in 1989 (Weiser et al., Proc. Natl. Acad. Sci. USA, 86: 7522 (1989)). The mouse macrophage migration inhibitory factor was then also cloned (Bernhagen et al., Nature, 365: 756 (1993)).
[0006]
Thereafter, various properties of macrophage migration inhibitory factor have been revealed. For example, macrophage migration inhibitory factor is a major component of secretory granules in adrenocorticotropic hormone-producing cells in the anterior pituitary gland. Macrophage migration inhibitory factors are also thought to play a central role in host reactions leading to endotoxin shock (Bernhagen et al., Nature, 365: 756 (1993)). In addition, macrophage migration inhibitory factor has post-translational glycosylation inhibitory activity of immunoglobulin E-binding factor and is regarded as a “glycosylation inhibitor” (Mikayama et al., Proc. Natl. Acad. Sci. U). S.A., 90: 10056 (1993)). Furthermore, expression of macrophage migration inhibitory factor mRNA has been observed in fetal eye lenses and fibroblasts activated by growth factors (Wistow et al., Proc. Natl. Acad. Sci. US). A., 90: 1272 (1993); Lanahan et al., Mol. Cell. Biol., 12: 3919 (1992)). Macrophage migration inhibitory factor is expressed in human melanoma cells and is involved in tumor cell proliferation and angiogenesis (Shimizu et al., Biochem. Biophys. Res. Com., 264: 751 (1999)). However, there has been no report on the physiological effect of macrophage migration inhibitory factor on stem cells.
[0007]
[Problems to be solved by the invention]
An object of the present invention is to provide a hematopoietic stem cell proliferating agent that can proliferate human hematopoietic stem cells and hematopoietic progenitor cells outside the body more efficiently than before. A further object of the present invention is to be used as a therapeutic agent for various hematopoietic organ diseases, cancer radiotherapy and hematopoietic failure during chemotherapy, to be used for mass production of blood cells, and to hematopoietic stem cells at the time of gene therapy. It is an object of the present invention to provide a hematopoietic stem cell proliferating agent that can be used for improving the efficiency of gene introduction and further used as a diagnostic agent and a test agent.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have found that a protein that activates the proliferation of hematopoietic stem cells exists in the culture supernatant of mouse myeloma cells (NS-1 cells). By separating, purifying, and identifying this protein, the present inventors have clarified that this protein is a macrophage migration inhibitory factor that is novel as a hematopoietic stem cell growth factor, and uses their hematopoietic stem cell proliferation activity. As a result, the present invention has been completed.
[0009]
The hematopoietic stem cell proliferating agent of the present invention contains a macrophage migration inhibitory factor. In one embodiment, the macrophage migration inhibitory factor is a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, wherein one or several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 1. It may be a polypeptide having an amino acid sequence and having hematopoietic stem cell proliferation activity, or a modified form thereof.
[0010]
In one embodiment, the macrophage migration inhibitory factor is a polypeptide having the amino acid sequence represented by SEQ ID NO: 2, wherein one or several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 2. It may be a polypeptide having an amino acid sequence and having hematopoietic stem cell proliferation activity, or a modified form thereof.
[0011]
The method for proliferating hematopoietic stem cells or hematopoietic progenitor cells of the present invention includes the step of proliferating hematopoietic stem cells or hematopoietic progenitor cells in a non-human animal or in vitro using any of the hematopoietic stem cell proliferating agents described above.
[0012]
In one embodiment, a foreign gene can be introduced into a hematopoietic stem cell or a hematopoietic progenitor cell in the expansion step.
[0013]
The method for proliferating human tissue stem cells or tissue progenitor cells of the present invention includes the step of proliferating human tissue stem cells or tissue progenitor cells in a non-human animal or in vitro using any of the hematopoietic stem cell proliferating agents described above.
[0014]
The diagnostic or testing agent for blood system diseases of the present invention contains any of the above hematopoietic stem cell proliferating agents.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
In practicing the present invention, protein separation and analysis methods and assay methods known in the art are employed unless otherwise indicated.
I. Definition
Hereinafter, terms used to describe the present invention will be described. Macrophage migration inhibitory factor is a lymphokine first discovered as a protein that inhibits migration of macrophages in guinea pigs as described above. In the present invention, the term “macrophage migration inhibitory factor” is not limited to so-called macrophage migration inhibitory factor polypeptides, polypeptides having substantial homology in amino acid sequence to these polypeptides, and these polypeptides Any modification of is also included. Examples of homologous polypeptides are species variants and allelic variants.
[0016]
The human-derived macrophage migration inhibitory factor includes the amino acid sequence of SEQ ID NO: 1 in the sequence listing. In the case of a macrophage migration inhibitory factor derived from a mouse, the amino acid sequence of SEQ ID NO: 2 in the sequence listing is included.
[0017]
It is clear that human-derived polypeptides are preferred for human disease or therapeutic purposes and in the proliferation of human hematopoietic stem cells. However, homologous polypeptides from other mammals can also be used depending on the purpose. Furthermore, comparison with other mammalian polypeptides is important in obtaining a variant that retains the desired activity of a human-derived polypeptide.
[0018]
The macrophage migration inhibitory factor used in the present invention is not necessarily limited by the specific sequence described above, and an amino acid sequence in which one or several amino acids are deleted, substituted or added to these sequences. A homologous polypeptide having a desired activity and having a desired activity (that is, a homologue of macrophage migration inhibitory factor) is also included. Here, the mutation of up to “several” amino acids is not necessarily intended to be restricted to a specific upper limit as long as the desired activity is obtained, but typically about 60 or less, preferably about It may be in the range of 40 or less, more preferably about 20 or less. Examples of amino acid additions include the addition of one amino terminal Met residue.
[0019]
Conservative substitution of amino acids is one of the preferred means for obtaining homologous polypeptides. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; Tyrosine.
[0020]
Sequence identity (homology) between two amino acid sequences is determined by introducing gaps if necessary to optimize residue fit. A polypeptide having substantial amino acid sequence homology to human macrophage migration inhibitory factor is typically at least about 60%, preferably at least about 70% compared to the amino acid sequence of human macrophage migration inhibitory factor, More preferably, it can be expressed as a polypeptide having at least about 80%, more preferably at least about 90% or more homology. Software for determining homology is easily available, for example, Gene Works (Intelligenetics Inc.).
[0021]
The modified form of macrophage migration inhibitory factor used in the present invention is a polypeptide having a sequence identical or homologous to the above sequence, the amino acid side chain or amino terminus or carboxyl terminus of which is modified, and the desired activity Are retained polypeptides. The macrophage migration inhibitory factor homologues and modifications are collectively referred to as functional equivalents. Examples of the above-mentioned modified examples include polypeptides in which the amino terminus is acylated (for example, acetylated), and polypeptides in which the carboxy terminus is amidated or esterified.
[0022]
In the present invention, “having hematopoietic stem cell proliferation activity” is defined as CD34 when measured under substantially the same conditions as in Example 16 below (the addition concentration of macrophage migration inhibitory factor is 20 ng / ml). The number of cells positive for the antigen and negative for the CD38 antigen (hereinafter referred to as CD34 + CD38−) is greater than the number of cells shown in the control group of Example 16, typically about 110% or more, preferably about 200. % Or more.
[0023]
“Hematopoietic stem cells” are pluripotent cells that have the potential to differentiate into all blood systems, including not only bone marrow cells such as red blood cells, white blood cells, and megakaryocytes, but also lymphoid systems such as T cells and B cells. Refers to cells that are capable of self-proliferation. Hematopoietic stem cells are characterized by being positive for CD34 antigen and negative for CD38 antigen (CD34 + CD38-). Even more immature hematopoietic stem cells are characterized by being positive for CD34 antigen and negative for CD38 antigen, CD33 antigen, CD19 antigen, and CD2 antigen (CD34 + CD38-33-19-2-). A “hematopoietic progenitor cell” refers to a cell that has been determined to differentiate into a specific cell lineage of the blood system, but can proliferate by division. Hematopoietic progenitor cells are characterized by being positive for both CD34 and CD38 antigens (CD34 + CD38 +).
[0024]
“Tissue stem cell” refers to a cell that exhibits the ability to self-renew as it divides, and at the same time has the ability to differentiate into cells with different properties. Hematopoietic stem cells, stem cells of the epidermal basement membrane, stem cells of the small intestinal mucosal epithelium, nervous system Includes all types of stem cells, including stem cells. “Tissue progenitor cells” refer to cells in the previous stage that differentiate and mature into a specific tissue.
II. Macrophage migration inhibitory factor with hematopoietic stem cell proliferation activity
In the present invention, macrophage migration inhibitory factor is used as an active ingredient of a hematopoietic stem cell proliferating agent. Macrophage migration inhibitory factors can be isolated from natural sources, produced using recombinant DNA technology, or chemically synthesized.
[0025]
When the macrophage migration inhibitory factor is isolated from a natural source, it can be purified, for example, as follows. First, myeloma cells (for example, P3-NSI / 1-Ag4-1 (abbreviation: NS-1) cells) are cultured, and hematopoietic stem cell proliferating activity protein is separated and purified from the obtained culture supernatant. “Myeloma cells” are cancerous cell lines that have the ability to proliferate permanently. Myeloma cells are known for a variety of uses. For example, Kohler and Milstein have sensitized mouse-derived myeloma cells, P3-X63-Ag8 (abbreviated as X63) cells, with sheep red blood cells. Cell fusion with spleen-derived lymphocytes produced a B cell hybridoma (Kehler et al., Nature, 256: 495 (1975)). X63 cells are selected from the cell populations of immunoglobulin G1 kappa chain-producing myeloma cells derived from BALB / c mice that are resistant to 8-azaguanine and cannot grow in a medium containing hypoxanthine, aminopterin and thymidine by cloning. The obtained cell line. NS-1 cells are myeloma cells derived from X63 cells, which are known to synthesize only the immunoglobulin G1 kappa chain but not to secrete it (Kehler et al., Eur. J. Immunology, 6: 511 (1976). )).
[0026]
The present inventors have surprisingly found that hematopoietic stem cell proliferating activity exists in this NS-1 cell culture supernatant. Therefore, the hematopoietic stem cell proliferating factor in the present invention can be isolated from the culture supernatant of NS-1 cells or the culture supernatant of other myeloma cells exhibiting similar activity. Separation and purification of the hematopoietic stem cell proliferation activity protein found in the myeloma cell culture supernatant starts by preparing a large amount of myeloma cell culture supernatant exhibiting hematopoietic stem cell proliferation activity. Cell culture may be performed in accordance with a normal cell line culture method. For example, in an animal cell culture medium containing an appropriate ratio of fetal calf serum (FCS), CO set at 37 ° C. 2 Incubate in an incubator.
[0027]
In order to separate and purify proteins from the culture supernatant, it is necessary to reduce contaminating proteins as much as possible. Therefore, it is preferable that the medium is replaced with a serum-free medium immediately before the increase in the content of hematopoietic stem cell proliferation active protein, and the culture is continued, and then the culture supernatant is collected. The collected culture supernatant can be passed through a filter to remove cell debris.
[0028]
In order to separate and purify hematopoietic stem cell proliferating protein from the obtained culture supernatant, a normal protein separation and purification method can be used. To concentrate the culture supernatant, an ultrafiltration membrane can be used. The concentrated culture supernatant is applied to a gel filtration column using HPLC, and a fraction having hematopoietic stem cell proliferation activity is collected. The resulting active fraction is applied to a reverse phase HPLC column. A fraction having hematopoietic stem cell proliferation activity is recovered from the eluted fraction. The fraction thus obtained has sufficient purity, and can be used for amino acid sequence analysis with a protein sequencer without further purification. Thus, hematopoietic stem cell proliferating protein found in the myeloma cell culture supernatant can be identified.
III. Confirmation of hematopoietic stem cell proliferation activity
Next, an example of a method for confirming the hematopoietic stem cell proliferation activity of the macrophage migration inhibitory factor will be described. Hematopoietic stem cells (CD34 + CD38-33-19-2-cells) prepared from umbilical cord blood are plated on microtiter plates. As a medium used for the proliferation of hematopoietic stem cells, in the test group, a medium (stem cell growth factor / interleukin 6 //) containing 15% FCS-RPMI1640 and the cytokines stem cell growth factor, interleukin 6, and interleukin 3. Interleukin 3 medium) is prepared, and a medium to which a sample containing a target polypeptide (for example, human macrophage migration inhibitory factor) to be assayed for hematopoietic stem cell proliferation activity is added is used. In the control group, a medium in which 2% FCS-RPMI1640 is added to the stem cell growth factor / interleukin 3 medium is used in place of the sample containing the target polypeptide. After culturing these at about 37 ° C. for about 10 to 30 days, the cells were treated with CD34-FITC antibody (CD34 antibody combined with fluorescein isothiocyanate dye), and the cells were taken up by FACScan for a certain period of time. To determine the CD34 + cell ratio, measure the viable cells by the trypan blue method, and calculate the CD34 + cell number by multiplying the number of viable cells by the CD34 + cell ratio. In this way, hematopoietic stem cell proliferation activity of the target polypeptide can be confirmed.
IV. Hematopoietic stem cell proliferating agent and use thereof
The hematopoietic stem cell proliferating agent of the present invention is provided as a composition containing a macrophage migration inhibitory factor having hematopoietic stem cell proliferating activity and an arbitrary medium that does not substantially inhibit the activity. Here, “macrophage migration inhibitory factor” is used in a broad sense and includes homologues and modified forms of the above-mentioned macrophage migration inhibitory factor in addition to normal macrophage migration inhibitory factor. Hematopoietic stem cell proliferating agents for human administration typically can contain any pharmaceutically acceptable excipient known to those of skill in the art in addition to an effective amount of macrophage migration inhibitory factor. Examples of excipients include lactose, corn starch, magnesium stearate, alum and the like.
[0029]
The hematopoietic stem cell proliferating agent of the present invention can be prepared according to a method known in the art. The hematopoietic stem cell proliferating agent of the present invention can be in any shape. The hematopoietic stem cell proliferating agent of the present invention can be a solid such as a tablet, pill, capsule, granule; or a liquid such as an aqueous solution and suspension. When the hematopoietic stem cell proliferating agent of the present invention is orally administered as a tablet, excipients such as lactose, corn starch, and magnesium stearate can be usually used. When the hematopoietic stem cell proliferating agent of the present invention is orally administered as a capsule, usually excipients such as lactose and dried corn starch can be used. For oral administration as an aqueous suspension, macrophage migration inhibitory factor may be used in combination with an emulsion or suspension. Aqueous suspensions may contain sweetening and flavoring agents as desired. When the hematopoietic stem cell proliferating agent of the present invention is injected intramuscularly, intraperitoneally, subcutaneously, and intravenously, a macrophage migration inhibitory factor is dissolved in a sterilized solution to prepare a buffer, and the pH is adjusted to an appropriate value. . When the hematopoietic stem cell proliferating agent of the present invention is administered intravenously, the proliferating agent is preferably isotonic.
[0030]
By using the hematopoietic stem cell proliferating agent of the present invention, it is possible to proliferate a large amount of tissue stem cells or tissue progenitor cells containing hematopoietic stem cells or hematopoietic progenitor cells. For example, blood cells can be mass-produced by growing hematopoietic stem cells or hematopoietic progenitor cells in vitro using the hematopoietic stem cell proliferating agent of the present invention. The effective amount of the hematopoietic stem cell proliferating agent and the conditions for production and recovery of blood cells can be appropriately selected by those skilled in the art.
[0031]
Furthermore, the hematopoietic stem cell proliferating agent of the present invention is used for the treatment of various hematopoietic organ diseases such as immunosuppressive disorders, and patients whose increase in blood cells is desired, such as cancer hematopoietic failure due to radiotherapy and chemotherapy. Can do. In therapeutic use, the hematopoietic stem cell proliferating agent of the present invention can be administered in the form of a conventional polypeptide formulation as described in Remington's Pharmaceutical Sciences, Mack Publishing Company (Easton, PA). For example, the hematopoietic stem cell proliferating agent of the present invention can be administered by parenteral administration such as oral administration, intravenous administration, intramuscular injection, intraperitoneal injection, and subcutaneous injection. These polypeptides can be supplemented into amniotic fluid. Preferably, these polypeptides can be administered by injection.
[0032]
When the hematopoietic stem cell proliferating agent of the present invention is administered to a human, the daily dose is usually determined by a person skilled in the art in consideration of patient symptoms, severity, individual differences in sensitivity, body weight, age, etc. Can be determined. The hematopoietic stem cell proliferating agent of the present invention may be administered once a day or divided into several times a day.
[0033]
In gene therapy, it is known that the efficiency of introducing a target gene depends on how many cells are dividing in the process of introducing the gene into the cell. Therefore, a very high gene transfer efficiency can be obtained by introducing a gene while proliferating hematopoietic stem cells or hematopoietic progenitor cells using the hematopoietic stem cell proliferating agent of the present invention.
[0034]
Furthermore, the hematopoietic stem cell proliferating agent of the present invention can be used as it is as a diagnostic agent and a diagnostic agent for blood system diseases (for example, leukemia, myeloma, anemia, etc.).
[0035]
【Example】
Hereinafter, the macrophage migration inhibitory factor as the hematopoietic stem cell proliferating agent in the present invention will be described more specifically.
[0036]
(Example 1: Preparation of hematopoietic stem cells from human umbilical cord blood)
Mononuclear from human umbilical cord blood (approx. 50 ml using heparin as an anticoagulant) donated from the Obstetrics and Gynecology Department by Ficoll method (Amersham Pharmacia Biotech, using Ficoll Pack) Spherical fractions were prepared and stored frozen. This was freeze-thawed at the start of the assay, washed with 15% FCS-RPMI1640 (manufactured by Nikken Biomedical Research Institute), and CD34-FITC antibody (CD34 antibody was bound with fluorescein isothiocyanate dye) CD38-PE antibody (CD38 antibody conjugated with phycoerythrin dye), CD33-PE antibody (CD33 antibody conjugated with phycoerythrin dye), CD19-PE antibody (CD19 antibody phycoerythris Each antibody (Immunotech) was treated with CD2-PE antibody (CD2 antibody bound with phycoerythrin dye), and hematopoietic was performed with FACStar (Becton Dickinson) Stem cell CD34 + CD38-33-19-2-cells were collected.
[0037]
(Example 2: Construction of hematopoietic stem cell growth factor detection system using hematopoietic stem cells of human umbilical cord blood)
Human umbilical cord blood CD34 + CD38-33-19-2-cells collected in Example 1 were plated on a 96-well U-type plate (manufactured by Coaster) (50 cells / 100 μl / well). The medium used was a 15% FCS-RPMI1640 supplemented with cytokines stem cell growth factor (25 ng / ml), interleukin 6 (20 ng / ml), and interleukin 3 (20 ng / ml). 20 μl (/ well) of sample to be measured for hematopoietic stem cell proliferation activity was added.
After liquid culture at 37 ° C. for 14 to 27 days, the number of viable cells was measured by the trypan blue method (trypan blue manufactured by Dainippon Pharmaceutical Co., Ltd.). A hemocytometer was used for cell counting.
The number of CD34 + cells and the number of CD34 + CD38− cells were calculated as follows. First, the cells after liquid culture were treated with CD34-FITC antibody (manufactured by Immunotech) and CD38-PE antibody (manufactured by Immunotech) (cell 10 Five Were stained with 50 ng antibody). After washing the stained cells twice with PBS (-) (phosphate-buffered saline, pH 7.2; manufactured by Nikken Biomedical Laboratories, Inc.) (FCS 1%, containing sodium azide 0.01%) Then, propidium iodide (manufactured by Sigma-Aldrich Japan) was added to a final concentration of 10 μg / ml to stain the cells. The stained cells were analyzed with a flow cytometer FACScan (manufactured by Becton Dickinson) (flow cytometry) to obtain a CD34 + cell ratio and a CD34 + CD38− cell ratio, and the number of live cells was multiplied by the ratio to obtain the number of CD34 + cells. And the number of CD34 + CD38− cells was calculated.
[0038]
(Example 3: Preparation of mouse myeloma cell culture and culture supernatant thereof)
Mouse myeloma cell (NS-1) culture is 1.0 × 10 cells. Five Suspended in 15 ml of 15% FCS-RPMI1640 medium to a concentration of 5 ml / ml, put in a culture dish (manufactured by Greiner) (diameter 10 cm), 5% CO 2 The culture was started at 37 ° C. in an incubator. Culture supernatants were collected on
[0039]
(Example 4: Preparation of NS-1 cell growth curve)
The number of live cells and the number of dead cells were measured by the trypan blue method on
[0040]
(Example 5: Determination of NS-1 cell culture supernatant recovery time)
NS-1 cells were cultured, and the culture supernatant collected on
[0041]
(Example 6: Examination of concentration dependency of hematopoietic stem cell proliferation activity of NS-1 cell culture supernatant)
The concentration dependence of the hematopoietic stem cell proliferation activity of the NS-1 cell culture supernatant collected in Example 3 was examined. Using a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells, the NS-1 cell culture supernatant on the 25th day of culture is kept undiluted, 2-fold diluted, 4-fold diluted, 8-fold diluted, 16-fold. Dilution, 32-fold dilution, 64-fold dilution, 128-fold dilution, and 256-fold dilution were added to evaluate hematopoietic stem cell proliferation activity. The numbers of CD34 + cells were 238, 144, 74, 104, 45, 1, 0, 11 and 10, respectively. The numbers of CD34 + CD38− cells were 107, 36, 44, 37, 17, 1, 0, 2, and 1, respectively. Thus, the growth of hematopoietic stem cells was almost dependent on the concentration of the NS-1 cell culture supernatant.
[0042]
(Example 7: Large-scale preparation of NS-1 cell culture supernatant)
NS-1 cells 1.0 × 10 Five / Ml in a 15% FCS-RPMI1640 medium 50 ml, culture flask (Greiner, 150 cm 2 ). 5% CO 2 Cultivation was started at 37 ° C. in an incubator, and the culture supernatant on the fifth day after the culture was collected, and the supernatant that passed through a 0.22 μm filter (manufactured by Millipore Japan) was collected. A total of 3,620 ml of culture supernatant was collected.
[0043]
(Example 8: Purification of NS-1 cell culture supernatant by gel filtration)
15 ml of the NS-1 cell culture supernatant collected in Example 7 was concentrated to 1 ml with an ultrafiltration filter (manufactured by Gelman Sciences) with a molecular weight cut off of 10,000, and the entire amount was separated by Sephacryl S-200 column (Amersham Pharmacia Biotech). (Made by company) (diameter 10 mm, height 19.1 cm). For the equilibration buffer, RPMI1640 medium (manufactured by Nissui Pharmaceutical) was used to collect the eluted fractions.
When the elution fraction was evaluated for hematopoietic stem cell growth activity using a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells constructed in Example 2, activity was observed in the fraction having a molecular weight of about 20,000.
A 15% FCS-RPMI1640 medium stored at 37 ° C. for 5 days was subjected to gel filtration using a Sephacryl S-200 column in the same manner, and the same hematopoietic stem cell growth factor detection system was evaluated. Was hardly recognized.
From the above, it was found that the hematopoietic stem cell growth activity factor is present in the culture supernatant fraction having a molecular weight of around 20,000.
[0044]
(Example 9: Purification of NS-1 cell culture supernatant by gel filtration fraction by reverse phase HPLC)
The active fraction obtained by gel filtration obtained in Example 8 was subjected to reverse phase HPLC. As the reverse phase HPLC column, YMC-Pack PROTEIN-RP (manufactured by YMC Co., Ltd., length 250 mm, inner diameter 4.6 mm) was used, and LC-10A manufactured by Shimadzu Corporation was used as the HPLC apparatus. Elution is trifluoroacetic acid (TFA) -acetonitrile (CH Three CN) system. That is, 0.1% TFA (pH 2.0) for solution A and 80% CH for solution B Three Using CN-0.1% TFA (pH 2.0), the concentration of solution B was 0% for the first 5 minutes, and the concentration of solution B was changed from 0% to 70% in the next 70 minutes. Similarly, elution was performed with a linear gradient from 70% to 100% in 10 minutes. When the eluted fraction was evaluated by a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells, activity was observed in the fraction at a retention time of about 60 minutes and the fraction at about 63 minutes.
[0045]
(Example 10: NS-1 cell culture by serum-free culture and preparation of culture supernatant thereof)
In order to produce a culture supernatant that is easier to purify than a medium containing 15% FCS, an NS-1 cell culture method by serum-free culture was established. NS-1 cells 1 × 10 Five Cultivation was started with 40 ml of 15% FCS-RPMI1640 medium at a concentration of 1 ml / ml in a dish (made by Greiner) having a diameter of 14.5 cm. On the 4th day of culture, the medium was replaced with 40 ml of fresh 15% FCS-RPMI 1640 medium, and further cultured for 1 day. Subsequently, the plate was washed twice with PBS (−), transferred to 40 ml of RPMI1640 medium without serum, and cultured in a new dish for 1 day. Next, the culture was passed through a 0.22 μm filter to remove cell debris, and the culture supernatant was collected. The collected culture supernatant was subjected to evaluation with a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells to confirm that there was activity.
[0046]
(Example 11: Purification of NS-1 cell serum-free culture supernatant by gel filtration)
2 ml of the concentrated culture supernatant obtained by concentrating 20 ml of the NS-1 cell serum-free culture supernatant collected in Example 10 to 1/10 with an ultrafiltration filter having a molecular weight cut off of 10,000 (manufactured by Gelman Sciences) , TSKgel G3000SW XL Applied to the column. As the equilibration buffer, a 50 mM phosphate buffer containing 0.3 M sodium chloride was used, and the eluted fractions were collected. When the eluted fraction was evaluated by a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells, activity was observed in the fraction having a molecular weight of about 1 to 20,000.
[0047]
(Example 12: Purification of NS-1 cell serum-free culture supernatant by gel filtration fraction by reverse phase HPLC)
14 ml of the active fraction obtained by gel filtration obtained in Example 11 was subjected to reverse phase HPLC. As the reverse phase HPLC column, YMC-Pack PROTEIN-RP (manufactured by YMC Co., Ltd., length 250 mm, inner diameter 4.6 mm) was used, and LC-10A manufactured by Shimadzu Corporation was used as the HPLC apparatus. Elution is TFA-CH Three Performed in a CN system. That is, 0.1% TFA (pH 2.0) for solution A and 80% CH for solution B Three Using CN-0.1% TFA (pH 2.0), the concentration of solution B was 0% for the first 15 minutes, and the concentration of solution B was changed from 0% to 70% in the next 70 minutes. Similarly, elution was performed with a linear gradient from 70% to 100% in 10 minutes.
CH Three Peaks with a CN concentration of around 47% (retention times of 71 minutes, 72 minutes, and 74 minutes in total 3) were collected, re-chromatographed on the same column, and purified in the same manner.
[0048]
(Example 13: Confirmation of amino terminal amino acid sequence of purified sample)
Three samples obtained by purification by rechromatography of YMC-Pack PROTEIN-RP in Example 12 above were applied to a protein sequencer (Applied Biosystems Sequencer: Model 492) and analyzed.
The one at 71 minutes matched mouse cyclophilin B by amino-terminal analysis of 25 amino acid residues. The 72 minute one was consistent with mouse cyclophilin A. The one at 74 minutes appeared to have a structure in which its main component protein cannot be degraded by Edman. However, a minor constituent protein coincided with mouse macrophage migration inhibitory factor. When a peptide map using tosylphenylalanyl chloromethyl ketone (TPCK) -trypsin was prepared and analyzed for a sample with a retention time of 74 minutes, the amino-terminal peptide could not be degraded by Edman. Was consistent with mouse cyclophilin A. This protein was considered a mouse cyclophilin A-like protein.
[0049]
(Example 14: Separation of mixture in sample with retention time of 74 minutes)
In Example 13, it was found that in the sample having a retention time of 74 minutes, the mouse macrophage migration inhibitory factor that is a minor constituent protein and the mouse cyclophilin A-like protein that is the main constituent protein were mixed. I tried to separate them. Several lots of NS-1 cell culture supernatant was purified by the method of Examples 10, 11 and 12, and the chromatographic pattern of reverse phase HPLC was examined. In some lots, the retention time reached a peak of 74 minutes. It was found that the corresponding peak was divided into two. Of these peaks, the retention time peak was analyzed with a protein sequencer. This peak was found to be a mouse macrophage migration inhibitory factor (FIG. 1). Similarly, peptide mapping using TPCK-trypsin and analysis with a protein sequencer also revealed that the peak with an earlier retention time is a mouse cyclophilin A-like protein.
[0050]
(Example 15: Confirmation of hematopoietic stem cell proliferation activity of purified sample)
Using the amplification of the number of immature hematopoietic stem cells CD34 + CD38− cells as an index, the activity of the mouse macrophage migration inhibitory factor fraction obtained in Example 14 was examined using the hematopoietic stem cell growth factor detection system constructed in Example 2. . As a control, 2% FCS-RPMI 1640 was used. Cells cultured in 5 wells of 96 wells were mixed, and the number of viable cells contained therein was measured by the trypan blue method described in Example 2. The calculation of the number of CD34 + cells and the number of CD34 + CD38− cells was also calculated by the method described in Example 2. The concentration of the mouse macrophage migration inhibitory factor fraction was not accurately determined, but was about 2 to 20 ng / ml.
As a result, the total number of living cells, the number of CD34 + cells, and the number of CD34 + CD38− cells were amplified as compared with the control group (typical results are shown in FIGS. 2 and 3). The total number of living cells was amplified 18.8 times compared to the control group, the number of CD34 + cells was 3.8 times that of the control group, and the number of CD34 + CD38− cells was amplified 3.4 times that of the control group. It was. This indicates that mouse cord macrophage migration inhibitory factor has human umbilical cord blood hematopoietic stem cell proliferation activity.
[0051]
(Example 16: Confirmation of hematopoietic stem cell proliferation activity of E. coli type human macrophage migration inhibitory factor)
Hematopoietic stem cells (CD34 + CD38− cells) prepared from human umbilical cord blood were plated in a 5-well on a 96-well U-type plate (200 cells / 100 μl / well). In the test group, the medium used for the proliferation of hematopoietic stem cells was 15% FCS-RPMI1640, stem cell growth factor (25 ng / ml), interleukin 6 (20 ng / ml), and interleukin 3 (20 ng / ml). ) Supplemented medium (stem cell growth factor / interleukin 6 / interleukin 3 medium), and a sample (human macrophage migration inhibitory factor) containing E. coli type human macrophage migration inhibitory factor (manufactured by R & D Systems). 2% FCS-RPMI1640 dissolved in 20 μl / well (the final concentration of human macrophage migration inhibitory factor was 20 ng / ml). In the control group, a medium in which 20 μl (/ well) of 2% FCS-RPMI1640 was added to the stem cell growth factor / interleukin 6 / interleukin 3 medium was used instead of the above-mentioned sample containing human macrophage migration inhibitory factor. This experiment was performed in triplicate.
After culturing at 37 ° C. for 14 days and 27 days, 5 wells were combined into one, and the number of viable cells contained therein was measured by the trypan blue method described in Example 2. The calculation of the number of CD34 + cells and the number of CD34 + CD38− cells was also calculated by the method described in Example 2. As a result, the number of viable cells, CD34 + cells, and CD34 + CD38− cells were clearly determined in the test group (human macrophage migration inhibitory factor added group) in comparison with the control group. The number of cells increased (FIGS. 4 and 5). In the case of FIG. 4, the total number of living cells is 25.3 times that of the control group, the number of CD34 + cells is 13.4 times that of the control group, and the number of CD34 + CD38− cells is 10.3 times that of the control group. Amplified 5 times. In the case of FIG. 5, the total number of viable cells is 21.3 times that of the control group, the number of CD34 + cells is 18.0 times that of the control group, and the number of CD34 + CD38− cells is 15.3 times that of the control group. Amplified twice. This indicates that the human umbilical cord blood hematopoietic stem cell proliferating activity exists in the Escherichia coli type human macrophage migration inhibitory factor.
[0052]
【The invention's effect】
According to the present invention, a hematopoietic stem cell proliferating agent containing a macrophage migration inhibitory factor is provided. This hematopoietic stem cell proliferating agent can be used as a therapeutic agent for various hematopoietic organ diseases, cancer radiotherapy and hematopoietic failure during chemotherapy, it can be used for mass production of blood cells, and gene for hematopoietic stem cells during gene therapy It can be used to improve the efficiency of introduction of and further utilized as a diagnostic agent and a test agent.
[0053]
[Sequence Listing]
[Brief description of the drawings]
FIG. 1 shows a purified chromatographic pattern of gel filtration fractions of NS-1 cell serum-free culture supernatant by reverse phase HPLC (PROTEIN-RP), in which the fraction of mouse macrophage migration inhibitory factor is clearly shown. FIG. FIG. 1 shows absorption at 214 nm on the vertical axis and reverse phase HPLC (PROTEIN-RP) retention time (minutes) on the horizontal axis. Absorption at 214 nm reflects the amount of protein contained in each fraction.
FIG. 2 is a flow cytometry diagram showing the expression status of CD34 antigen and CD38 antigen in cells that have been cultured for 15 days using a mouse macrophage migration inhibitory factor fraction purified from serum-free culture supernatant of NS-1 cells. Indicated by The vertical axis represents the fluorescence intensity of the CD38-PE antibody, and the horizontal axis represents the fluorescence intensity of the CD34-FITC antibody. The left side of the figure is a flow cytometry diagram of cells cultured and amplified using a control group (2% FCS-RPMI1640) and the right side using a macrophage migration inhibitory factor. Dots indicate cells. The cross line shows the vertical axis and the horizontal axis drawn on the flow cytometry diagram obtained by treating the cells cultured and amplified as described above with mouse immunoglobulin G1-FITC antibody and mouse immunoglobulin G1-PE antibody. A line on the axis separating each positive and negative is shown. That is, the lower right quadrant indicates CD34 + CD38−, the upper right quadrant indicates CD34 + CD38 +, the upper left quadrant indicates CD34−CD38 +, and the lower left quadrant indicates CD34−CD38−. It can be seen that the total number of viable cells, the number of CD34 + cells, and the number of CD34 + CD38− cells are clearly amplified as compared with the control group.
FIG. 3 is a diagram summarizing the human umbilical cord blood hematopoietic stem cell proliferating activity of a fraction of a mouse macrophage migration inhibitory factor purified from NS-1 cell serum-free culture supernatant. The vertical axis represents the number of cells (number), and the horizontal axis represents the live cells on the 15th day, the CD34 + cells on the 15th day, and the CD34 + CD38− cells on the 15th day in order from the left. The black column represents the control group, and the white column represents the number of cells when cultured using a macrophage migration inhibitory factor. The total number of living cells was amplified 18.8 times compared to the control group, the number of CD34 + cells was 3.8 times that of the control group, and the number of CD34 + CD38− cells was amplified 3.4 times that of the control group. In other words, the mouse macrophage migration inhibitory factor has human umbilical cord blood hematopoietic stem cell proliferation activity.
FIG. 4 is a summary of human umbilical cord blood hematopoietic stem cell proliferation activity (culture day 14) of E. coli type human macrophage migration inhibitory factor. The vertical axis indicates the number of cells (cells), and the horizontal axis indicates the live cells on the 14th day, the CD34 + cells on the 14th day, and the CD34 + CD38− cells on the 14th day in order from the left. The vertical bars indicate the standard deviation of each. The black column represents the control group, and the white column represents the number of cells when cultured using a macrophage migration inhibitory factor. The total number of viable cells was amplified 25.3 times compared to the control group, the number of CD34 + cells was 13.4 times compared to the control group, and the number of CD34 + CD38− cells was amplified 10.5 times compared to the control group. This indicates that the human umbilical cord blood hematopoietic stem cell proliferating activity exists in the Escherichia coli human macrophage migration inhibitory factor.
FIG. 5 is a summary of human umbilical cord blood hematopoietic stem cell proliferation activity (cultured on day 27) of E. coli type human macrophage migration inhibitory factor. The vertical axis represents the number of cells (cells), and the horizontal axis represents the living cells on the 27th day, the CD34 + cells on the 27th day, and the CD34 + CD38− cells on the 27th day in order from the left. The vertical bars indicate the standard deviation of each. The black column represents the control group, and the white column represents the number of cells when cultured using a macrophage migration inhibitory factor. The total number of viable cells was amplified 21.3 times compared to the control group, the number of CD34 + cells was 18.0 times compared to the control group, and the number of CD34 + CD38− cells was amplified 15.2 times compared to the control group. This indicates that the human umbilical cord blood hematopoietic stem cell proliferating activity exists in the Escherichia coli human macrophage migration inhibitory factor.
Claims (5)
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