JP3931353B2 - Wound healing agent - Google Patents

Description

ただし、２４２番目の Val は Ala に置換されていてもよい。
【００２１】
本発明の第４の態様は、ＴＭ様蛋白質とヒト細胞成長因子とを含有することを特徴とする細胞増殖および／または細胞成長促進剤である。好ましくは、肉芽組織形成促進剤である。ここで、ＴＭ様蛋白質としては、上述の第３の態様と同様に、上述のアミノ酸配列１を有するものが好ましい。また、ヒト細胞成長因子は、ヒトＩＧＦ−１、ヒトインスリン、ヒトＥＧＦ又はヒトｂ−ＦＧＦであることが好ましい。
【００２２】
ＴＭ様蛋白質とヒト細胞成長因子との組み合わせとしては、上述のアミノ酸配列１を有するＴＭ様蛋白質と、ヒトＩＧＦ−１、ヒトインスリン、ヒトＥＧＦおよびヒトｂ−ＦＧＦの少なくとも一つとの組み合わせが好ましい。
【００２３】
以下、本発明を詳細に説明する。
【００２４】
本明細書において、「ＴＭ様蛋白質」の語は、ＴＭおよびＴＭの生物学的もしくは免疫学的活性（即ち、トロンビンと結合し、トロンビンの凝固促進作用を抑制するとともにトロンビンのプロテインＣ活性化を著しく促進させるという活性）に必要な一部分のアミノ酸配列を有するペプチド又はそれらの類似物を意味する。ここでいう「ペプチド」の語は、換言すれば、後述するように、ヒトＴＭを構成する完全長の５５７アミノ酸残基の中の、ＴＭおよびＴＭの生物学的もしくは免疫学的活性を示すペプチドフラグメントを意味する。そして、「それらの類似物」の語は、前述のペプチドフラグメントのＯ−グリコシド結合糖鎖あるいはＮ−グリコシド結合糖鎖などの糖鎖を修飾あるいは欠失させたものを意味する。また、「創傷」の語は、外力によって生じた組織損傷を総括していうが、皮膚や粘膜面の連続性が離断した解放性損傷を創、連続性が保持された閉鎖性損傷を傷として区別して用いられることがあり、「創傷治癒」は、炎症反応が起こり、毛細血管等の透過性亢進や白血球等の遊走が起こって細菌や壊死物質が貪食除去されると共にフィブリン沈着等が起こる滲出破壊相、毛細血管の新生や線維芽細胞の増殖が始まり、肉芽が出現し、フィブリンが吸収されてコラーゲンに置き換わってゆく繊維増殖相および新生血管や滲出細胞が減少し始め、コラーゲン量が増加して創の収縮が起きる成熟期の機転をとる［南山堂医学大辞典、南山堂，１１５６−１１５７頁，１９９０］とされ、本発明においてもこれらの意味を包含する。肉芽（組織）とは、盛んに増殖している若い結合組織のことで、創傷の壊死性組織を吸収し、欠損部を埋め、線維化をおこす創傷治癒に際して形成される重要な組織である。線維芽細胞が肉芽組織の基本的構成細胞である。また、「創傷治癒剤」の語は、種々の創傷、例えば、角膜潰瘍、放射線角膜傷、角膜移植その他の手術誘発眼内創傷のような眼創傷、および切開、やけど、切り傷、破傷、深部術創、皮膚移植によるドナー部位創および潰瘍（皮膚、とこずれ、静脈うっ血および糖尿病）のような皮膚創のごとき上皮損傷、あるいは消化管の潰瘍、口腔・気道または尿路のごとき粘膜の損傷など、さらに、乾せん、日焼け、皮膚発疹のような皮膚障害等、の治癒を促進するための治療剤を広く包含する。
【００２５】
本発明に用いるＴＭ様蛋白質は、天然型あるいは遺伝子工学的に生産されたもののいずれでも良く、遺伝子工学的手法により得られる改変型あるいはキメラ型であっても良い。医薬品とする場合、好ましくはヒト由来のＴＭが望まれる。また、ヒト由来のＴＭは膜結合性であり、完全長のアミノ酸配列を持つものは水に対する溶解性が低い。このため、本発明においては完全長のアミノ酸配列を持たず、溶解性の高いＴＭ（以下、可溶性ＴＭと略する）がさらに好ましい。
【００２６】
このようなＴＭは、天然型については、例えば、ヒト胎盤由来のＴＭ［特開昭６０−１９９８１９］があげられる。また、可溶性ＴＭとしては、非還元状態での分子量が５５，０００〜５８，０００および６０，０００〜６５，０００のヒト尿由来可溶性ＴＭ［特開平３−８６９００］および非還元状態での分子量が７２，０００±３，０００および７９，０００±３，０００のヒト尿由来可溶性ＴＭ［特開平３−２１８３９９］が挙げられる。これらの内、分子量が７２，０００±３，０００および７９，０００±３，０００のヒト尿由来可溶性ＴＭが好ましい。これらヒト尿由来可溶性ＴＭは、特開平３−２１８３９９号公報に記載されているように、それぞれ、前述の本発明で好ましく使用することができるＴＭ様蛋白質の特徴１または特徴２を有している。
【００２７】
また、本発明に用いるＴＭ様蛋白質は、遺伝子組み替え技術で生産することができる。ヒトの治療剤に用いるためには、ヒトのアミノ酸配列を有するものが好ましい。具体的には、遺伝子工学的手法により製造され、鈴木らにより示されたヒトＴＭの一次構造［エンボ ジャーナル（ＥＭＢＯ．Ｊ．）６，１８９１−１８９７，１９８７；バイオケミストリー（Ｂｉｏｃｈｅｍ．）２６，４３５０−４３５７，１９８７］を有するポリペプチドおよびその生物学的もしくは免疫学的活性に必要な一部分のアミノ酸配列からなるフラグメントが挙げられる。
【００２８】
鈴木らによれば、完全長のヒトＴＭは５５７アミノ酸残基により構成され、それは、Ｎ末端より順に、Ｄ１、Ｄ２、Ｄ３、Ｄ４およびＤ５の５つのドメイン構造から構成される。また、その抗凝固活性は６つの上皮細胞成長因子（ＥＧＦ）様構造の繰り返しから成るＤ２ドメインの、４番目から６番目のドメイン（Ｅ４５６）に限局することが報告されている。本発明の治癒剤に使用し得るフラグメントの例としては、鈴木らにより開示されたＥ４５６ペプチド構造を包含する種々のペプチドフラグメント［特開平１−６２１９］あるいは名和らにより示された可溶性ＴＭ様蛋白質［バイオケミカル アンド バイオフィジカル リサーチコミュニケーションズ（Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．）１７１，７２９−７３７，１９９０］などが挙げられる。さらに具体的には、Ｄ２ドメインのＥＧＦ様構造の１番目から６番目までのドメイン構造（以下、Ｅ１２３４５６とする）を包含する、ヒトＴＭのＮ末端より２１４−４６６番目のアミノ酸残基により構成されるペプチド（以下、ＴＭＥ_1-6 とする）、１−４９１番目のアミノ酸残基により構成されるペプチド（以下、ｒｓＴＭとする）、２２７−４６２番目のアミノ酸残基により構成されるペプチド（Ｄ２）、１−４９７番目のアミノ酸残基により構成されるペプチド（以下、Ｄ１２３とする）、１−４６２番目のアミノ酸残基により構成されるペプチド（Ｄ１２）、２２７−４９７番目のアミノ酸残基により構成されるペプチド（Ｄ２３）、１−５５７番目のアミノ酸残基により構成されるペプチド（Ｄ１２３４５）、３４５−４６２番目のアミノ酸残基により構成されるペプチド（Ｅ４５６）などが好ましい。そして、これらのフラグメントの内、ＴＭの第２番目のドメイン、すなわちＥＧＦ様構造ドメインの１番目から６番目までの構造を包含する、２１４−４６６番目のアミノ酸残基により構成されるフラグメント（ＴＭＥ_1-6 )、即ち、前述のアミノ酸配列１を有するもの、ｒｓＴＭあるいはＤ１２３が最も好ましい。
【００２９】
なお、上記フラグメントは遺伝子組み替え技術を用いて、ＴＭ又は上記ペプチドの構造の一部を欠失させたり、アミノ酸を他のアミノ酸で置換あるいは修飾した改変体およびＯ−グリコシド結合糖鎖あるいはＮ−グリコシド結合糖鎖などの糖鎖を修飾あるいは欠失させた類似物などでも良い。具体的には、上記ポリペプチドの構成アミノ酸が一部欠如している［ＷＯ９２／００３２５、特開平２−２５５６９９、ＥＰ０４７４２７３Ａ２］か、他のアミノ酸に置換されたもの［ＷＯ９１／１５５１４、ＷＯ９３／１５５７５５、特開平５−３１０７８７］が挙げられる。また、メチオニンを他のアミノ酸に置換することによって酸化を防止したもの［ＷＯ９１／１５５１４］、あるいはアミノ酸配列を修飾することによって蛋白分解酵素による分解を防止したもの［ＷＯ９３／１５７５５］が挙げられる。また、コンドロイチン及び／又はコンドロイチン硫酸を含む糖鎖を有するもの［ＷＯ９１／０４２７６、ＷＯ９１／０５８０３、ＥＰ０４１２８４１Ａ１］、ウシトロンボモジュリン由来の酸性アミノ酸配列を含むＯ−グリコシド糖鎖結合部位を付加し、コンドロイチン硫酸糖鎖を結合させたもの［ＥＰ０４８８３１７Ａ２］、あるいは、Ｏ−グリコシル化部位領域の糖鎖を修飾あるいはＯ−グリコシル化部位領域を欠失させたもの［ＷＯ９２／０３１４９］、コンドロイチン及び／又はコンドロイチン硫酸を含まないもの［特開平４−２１０７００］などが挙げられる。
【００３０】
本発明第２の態様ならびに第４の態様は、以上のようなＴＭ様蛋白質の他にヒト細胞成長因子も有効成分として含有するものである。これにより、ＴＭ様蛋白質又はヒト細胞成長因子単独から創傷治癒剤ならびに細胞増殖および／または細胞成長促進剤を構成した場合に対して、大きな治癒効果ならびに促進効果を得ることが可能となる。
【００３１】
本発明において、ヒト細胞成長因子としては、ヒトＩＧＦ−１、ヒトインスリン、ヒトＥＧＦ、ヒトｂ−ＦＧＦの中から選ばれる１種を使用するか、あるいは複数種を併用することが好ましい。
【００３２】
また、本発明においては、細胞成長因子として上述のようにヒトＩＧＦ−１、ヒトインスリン、ヒトＥＧＦ、ヒトｂ−ＦＧＦを含有することができるが、これらに限定されない。それぞれ、ＩＧＦ、インスリン、ＥＧＦ、ＦＧＦ様活性を有する物質であればいずれでも良い［蛋白質核酸酵素，３６巻，７号，１０７８−１０８７頁，１２３７−１２４６頁，１９９１、臨床免疫，２７巻，サプルメント（Ｓｕｐｐｌ．）１６，３０７−３１６頁，１９９５］。例えば、動物の体内や動物細胞で産生される天然由来の物質、遺伝子組み替え技術で生産される物質、また遺伝子組み替え技術で、これらの構造の一部を欠失させたり、アミノ酸を他のアミノ酸で置換あるいは修飾した改変体および糖鎖を修飾あるいは欠失させた類似物などが挙げられる。好ましくは、ヒト由来かヒト由来と同等構造を有するものがよい。これらのいくつかは市販品として入手することができる。また、このようなヒト細胞成長因子としては、公知のいかなる製剤学的製造法により得られたものでも使用することができる。
【００３３】
本発明の第１の態様及び第２の態様のいずれの治癒剤ならびに本発明の第３の態様及び第４の態様のいずれの細胞増殖および／または細胞成長促進剤も種々の剤型にすることができ、その場合に種々の添加剤等の成分を配合することができる。例えば、ＴＭ様蛋白質は、薬剤として一般的に用いられる適当な担体または、媒体、例えば滅菌水や生理食塩水、植物油（例、ゴマ油、オリーブ油）、鉱油（例、ワセリン、パラフィン）、高級アルコール（例、セタノール、ステアリルアルコール）、高級脂肪酸（例、ステアリン酸、パルミチン酸）、無害性有機溶媒（例、エタノール）等、さらには必要に応じて賦形剤（例、マンニトール、カルボキシメチルセルロース）、着色剤、乳化剤（例、コレステロール）、懸濁剤（例、アラビアゴム）、界面活性剤（例、ポリオキシエチレン硬化ヒマシ油系界面活性剤、ポリエチレングリコール系界面活性剤）、溶解補助剤（例、界面活性剤、リン酸ナトリウム、酢酸ナトリウム）、吸着防止剤（例、非イオン性界面活性剤、ゼラチン）、安定化剤（例、アミノ酸、糖、糖アルコール、アルブミン）または保存剤（例、ベンジルアルコール、パラベン）、保湿剤（例、尿素、コレステロール）、酸化防止剤（例、トコフェロール）等と適宜組み合わせて、生体に効果的に投与するのに適した注射剤、経皮吸収剤、点眼剤、経鼻吸収剤、経口剤等の医薬用製剤、好ましくは注射剤もしくは経皮吸収剤に調製することが出来る。例えば、注射剤の製剤としては、凍結乾燥品や、注射用水剤、あるいは浸透圧ポンプに封入した形等で提供できる。また、経皮吸収剤としては、液剤、軟膏剤、クリーム剤、ゲル剤、バップ剤、人工皮膚類等で提供できる。
【００３４】
本発明の創傷治癒剤ならびに細胞増殖および／または細胞成長促進剤の投与方法としては、例えば、静脈内注射、皮下注射、筋肉内注射、経口投与等の方法によって、ＴＭ様蛋白質量として一日約０．０１〜１０００μｇ／ｋｇ、さらに好ましくは、０．１〜１００μｇ／ｋｇとなる量を投与したり、浸透圧ポンプ等に封入し生体に留置することにより連続的に投与することができる。なお、ＴＭ様蛋白質に加えてヒトＩＧＦ−１またはヒトインスリンまたはヒトＥＧＦまたはヒトｂ−ＦＧＦを有効成分として含有させた製剤は、上記のＴＭ様蛋白質投与と同時に、各因子をそれぞれ、一日約０．０１〜１０００μｇ／ｋｇ、さらに好ましくは、０．１〜１００μｇ／ｋｇとなる量を投与することが好ましい。
【００３５】
また、経皮吸収剤として、熱傷、創傷部位に、液剤、軟膏剤、クリーム剤、ゲル剤、バップ剤等の方法により局所適用することにより、例えば、ＴＭ様蛋白質濃度を約０．００１〜１０００μｇ／ｍｌ、さらに好ましくは、０．０１〜１００μｇ／ｍｌの濃度で適用することができる。なお、ヒトＴＭ様蛋白質に加えてヒトＩＧＦ−１またはヒトインスリンまたはヒトＥＧＦまたはヒトｂ−ＦＧＦを有効成分として含有する製剤は、上記のＴＭ様蛋白質適用と同時に、各因子をそれぞれ、一日約０．０１〜１０００μｇ／ｍｌ、さらに好ましくは、０．１〜１００μｇ／ｍｌとなる量を適用する。
【００３６】
さらに、熱傷の時に用いる培養皮膚移植等に際しては、細胞の培養液中に、例えば、ヒトＴＭ様蛋白質濃度を約０．００１〜１０００μｇ／ｍｌ、さらに好ましくは、０．０１〜１００μｇ／ｍｌの濃度で添加することもできる。なお、ＴＭ様蛋白質に加えてヒトＩＧＦ−１またはヒトインスリンまたはヒトＥＧＦまたはヒトｂ−ＦＧＦをＴＭ様蛋白質添加と同時に、各因子をそれぞれ、約０．０１〜１０００μｇ／ｍｌ、さらに好ましくは、０．１〜１００μｇ／ｍｌの濃度で添加することもできる。
【００３７】
【作用】
本発明の創傷治癒剤は、有効成分として、線維芽細胞などの細胞の成長および増殖促進効果を発揮するＴＭ様蛋白質、あるいはＴＭ様蛋白質とヒト細胞成長因子の双方を含有するので、種々の創傷、例えば、角膜潰瘍、放射線角膜傷、角膜移植その他の手術誘発眼内創傷のような眼創傷および切開、やけど、切り傷、破傷、深部術創、皮膚移植によるドナー部位創および潰瘍（皮膚、とこずれ、静脈うっ血および糖尿病）のような皮膚創のごとき上皮損傷、あるいは消化管の潰瘍、口腔・気道または尿路のごとき粘膜の損傷などの治療に用いることができる。さらに、乾せん、日焼け、皮膚発疹のような皮膚傷害にも用いることができる。
【００３８】
本発明の細胞増殖および／または細胞成長促進剤は、特に線維芽細胞の増殖／成長を促進し、肉芽組織形成促進に働く。また、細胞培養系に添加することにより、細胞の増殖を促進することができる。移植に用いるヒト細胞の培養液に添加してそのヒト細胞を培養することもできる。
【００３９】
【実施例】
以下にＴＭ様蛋白質の取得例及び本発明の実施例を挙げて、本発明をより具体的に説明するが、本発明に用いるＴＭ様蛋白質は、以下の取得例にあげたものに限定されることはなく、また、本発明は以下の実施例に限定されるものでもない。
【００４０】
取得例１（ヒトＴＭ様蛋白質の取得例：ＴＭＥ_1-6 )
名和らの方法［バイオケミカル アンド バイオフィジカル リサーチ コミュニケーションズ（Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．）１７１、７２９−７３７、１９９０］に準じて、ｐＲＳ７ＴＭを調製した。すなわち、ヒトｃＤＮＡライブラリーより得たＴＭ遺伝子のフラグメントをｐＵＣ１１９発現ベクターに挿入した後、制限酵素処理を行いｐＲＳ７ＴＭを調製した。さらに、ｐＲＳ７ＴＭのＴＭシグナルシーケンスをインターフェロンγシグナルシーケンスで置き換え、さらにＮ末端シーケンスを欠失させた。すなわち、Ｓａｌ−ＢｓｔＮ１制限酵素処理により８０ｂｐの新しいシグナルシーケンスを含むフラグメントを、ＰｓｔＩ制限酵素処理により１番目のＥＧＦドメインをコードしているフラグメントを取得した後、リンカーと結合させｐＴＭＰ２２を作製した。このｐＴＭＰ２２をＨｉｎｄＩＩＩ−ＭｌｕＩ制限酵素処理し、生じたフラグメントをｐＴＭｓ０７のＨｉｎｄＩＩＩ−ＭｌｕＩフラグメントと置換しｐＰ７ＴＭを作製した。Ｓｅｒ／Ｔｈｒ ｒｉｃｈ領域を欠失させるためにｐＴＭｓ０７をＨｉｎ ｃＩＩ制限酵素処理してフラグメントを単離し、さらにＨｐａＩＩ制限酵素処理を行うことにより６番目のＥＧＦドメインをコードしている７０ｂｐのフラグメントを取得した。このフラグメントのＬｙｓ−４６６の後にストップコドンを含むリンカーを結合させた後、Ａｐａ Ｉ−Ｘｂａ Ｉ制限酵素処理を行い、生じたフラグメントをｐＰ７ＴＭのＡｐａ Ｉ−Ｘｂａ Ｉ部位に組み込みｐＰＧ３ＴＭを作製した。
【００４１】
このプラスミド（ｐＰＧ３ＴＭ）を、常法に従ってチャイニーズハムスター卵母細胞株（ＣＨＯ−Ｋ１細胞株）にトランスフェクトした。この細胞を選択培地でさらに２〜３週間培養することにより安定した形質転換細胞を選抜し、生じたコロニーをさらに培養した。この培養上清を抗ＴＭモノクローナル抗体カラムとゲル濾過により精製し、ＴＭＥ_1-6 を得た。
【００４２】
取得例２（可溶性ＴＭの取得例：ＵＴＭ０）
特開平３−２１８３９９号公報に記載された方法に準じて調製した。すなわち
、原尿１００Ｌをアクリル繊維で濾過して尿中のウロキナーゼを吸着除去し、通過尿を限外濾過膜を使用して脱塩濃縮した。次いで、ＤＥＡＥセルロース（ワットマン社製）カラム、ＤＩＰ−トロンビン−アガロースクロマトグラフィーおよびセファクリルＳ−３００（ファルマシアファインケミカル社製）カラムを用いて順次精製し、活性画分を採取した（以下、ＵＴＭ０とする）。この画分は一晩蒸留水に対して透析した後凍結乾燥した。
【００４３】
取得例３（可溶性ＴＭの取得例：ｒｓＴＭ）
名和らの方法［バイオケミカル アンド バイオフィジカル リサーチ コミュニケーションズ（Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．）１７１、７２９−７３７、１９９０］に準じて、ｐＲＳ７ＴＭを調製した。すなわち、ヒトｃＤＮＡライブラリーより得たＴＭ遺伝子のフラグメントをｐＵＣ１１９発現ベクターに挿入した後、制限酵素処理等を行い、ｒｓＴＭをコードするプラスミドｐＴＭｓ０７を調製した。さらに、Ｎｅｏ^r 遺伝子を組み込み、発現プラスミドｐＴＭｓ０７−ｎｅｏを作製した。
【００４４】
このプラスミド（ｐＴＭｓ０７−ｎｅｏ）を、常法に従ってチャイニーズハムスター卵母細胞株（ＣＨＯ−Ｋ１細胞株）にトランスフェクトした。この細胞を選択培地でさらに２〜３週間培養することにより安定した形質転換細胞を選抜し、生じたコロニーをさらに培養した。この培養上清を抗ＴＭモノクローナル抗体カラムとゲル濾過により精製し、ｒｓＴＭを得た。
【００４５】
取得例４（可溶性ＴＭの取得例：Ｄ１２３）
ＷＯ９２／００３２５号公報の方法に準じて製造した。すなわち、ヒト胎盤ｃＤＮＡライブラリーより釣り上げたＤＮＡを利用してアミノ末端のアミノ酸配列がＡｌａ−Ｐｒｏ−Ａｌａ−であるアミノ酸４９７残基よりなる可溶性トロンボモジュリンを発現するベクターを調製し、これをＣＨＯ細胞に組み込んだ後、遺伝子増幅を行って高発現株を得た。この高発現株の培養液をＤＩＰ−トロンビン−アガロースカラムとゲル濾過により精製し、Ｄ１２３を得た。
【００４６】
実施例１（ヒトＴＭによる線維芽細胞に対する効果１：増殖促進効果）
スイス３Ｔ３細胞（Ｊａｐａｎｅｓｅ Ｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ Ｂａｎｋ）を１０％牛胎児血清（以下、ＣＳと略す。：ギブコ社）を含むＤＭＥＭ培地（ギブコ社）に懸濁し、３５ｍｍの培養プレートに１ウェルあたり１×１０ ^４ 個の密度で播種し、３７℃、５％ＣＯ_２／９５％Ａｉｒの条件で培養した。２４時間後に５０ｎｇ／ｍｌのヒトＴＭを含む種々の濃度のＣＳを含むＤＭＥＭ培地（０．１〜１０％ＣＳ）に培地交換し、その後は２日間に一度（図１の矢印参照）培地交換した。細胞は０．２５％ｔｒｙｐｓｉｎ／０．０２％ＥＤＴＡで処理し、細胞数を測定した。なお、ヒトＴＭは、取得例１に記載した方法に従い、ヒトＴＭのＥ１２３４５６を包含するペプチド（ＴＭＥ1-6 ）をチャイニーズハムスター卵母細胞（ＣＨＯ−Ｋ１細胞）で発現後、均一に精製したものを使用した。
【００４７】
この結果を図１に示す。図１から明らかなように、ＴＭＥ_1-6 は０．１％あるいは１％血清の存在下で細胞増殖作用を示した。また、ＵＴＭ０、ｒｓＴＭおよびＤ１２３も同様の作用を示す。
【００４８】
一般に、ｉｎ ｖｉｔｒｏでの増殖促進能は、ｉｎ ｖｉｖｏでの組織の成長を起こし得る細胞分化と直接関連しており、強いｉｎ ｖｉｔｒｏ増殖促進能を有する物質がｉｎ ｖｉｖｏにおいても成長刺激効果を有することは成長因子の研究においては良く知られているところである。従って、ヒトＴＭ様蛋白質は、生体でも効果を有することがわかる。
【００４９】
実施例２（ヒトＴＭによる線維芽細胞に対する効果２：ＤＮＡ合成促進効果）
スイス３Ｔ３細胞を１０％ＣＳを含むＤＭＥＭ培地に懸濁し、９６ウェルのマイクロタイタープレートに播種し、３７℃、５％ＣＯ₂ ／９５％Ａｉｒの条件でコンフルエントになるまで増殖させた。次いで、培養液を０．１％ＣＳを含む（又は含まない）ＤＭＥＭ培地で置換し４８時間培養することにより、細胞は完全に非活動性となった。次に、表１及び図２に示すように、種々の濃度のヒトＴＭおよび種々の濃度の成長因子を添加し、３７℃、２０時間培養した。その後³Ｈ-チミジン（１μＣｉ／ウェル）を加え、さらに８時間培養した。ウェルを氷冷リン酸緩衝液（以下、ＰＢＳと略す）で洗浄して、過剰の³Ｈ-チミジンを除いた後、細胞を０．２５％ｔｒｙｐｓｉｎ／０．０２％ＥＤＴＡで処理してプレートより剥離させ、グラスファイバーフィルター上に集めた。細胞を氷冷したトリクロル酢酸で洗浄後、フィルター上に残存する放射能を液体シンチレーションカウンターにより測定した。なお、ヒトＴＭは、取得例１に従い、ヒトＴＭのＥ１２３４５６を包含するペプチド（ＴＭＥ_1-6 ）をチャイニーズハムスター卵母細胞（ＣＨＯ−Ｋ１細胞）で発現後、均一に精製したもの、取得例２に従い精製したヒト尿由来可溶性ＴＭ（ＵＴＭ０）および取得例３に従い精製したｒｓＴＭを使用した。ＩＧＦ−１およびｂＦＧＦはインタージェン社、インスリンは和光純薬、ＥＧＦはアップステートバイオテクノロジー社よりそれぞれ購入して使用した。
【００５０】
結果を図２、図３および表１に示す。
【００５１】
図２から明らかなように、ＴＭＥ_1-6 は血清の存在下あるいは非存在下においてＤＮＡ合成促進作用を示した。また、図３から明らかなように、ＴＭＥ_1-6 、ｒｓＴＭおよびＵＴＭ０いずれもＤＮＡ合成促進作用を示し、ＵＴＭ０が最も強い作用を示した。
【００５２】
【表１】

表１から明らかなように、ＴＭＥ_1-6 は単独でＤＮＡ合成促進作用を示すのみならず、細胞成長因子として知られているＥＧＦやｂ−ＦＧＦとは相加的な作用を示し、インスリンあるいはＩＧＦ−１とは相乗的なＤＮＡ合成促進作用を示した。
また、ＵＴＭ０、ｒｓＴＭおよびＤ１２３も同様の作用を示した。
【００５３】
実施例３（ヒトＴＭによる線維芽細胞に対する効果３：グルコース輸送促進効果）
浜中らの方法［エクスペリメンタル セル リサーチ（Ｅｘｐ．Ｃｅｌｌ Ｒｅｓ．）１８６、８３−８９、１９９０］に準じて実施した。すなわち、スイス３Ｔ３細胞を１０％ＣＳを含むＤＭＥＭ培地に懸濁し、２４ウェルのマルチプレートに播種し、３７℃、５％ＣＯ₂ ／９５％Ａｉｒの条件でコンフルエントになるまで増殖させた。次いで、培養液を０．５％ＣＳを含むＤＭＥＭ培地で置換し４８時間培養した後、５０ｎｇ／ｍｌのヒトＴＭ、１０ｎｇ／ｍｌＥＧＦあるいは１０％ＣＳを添加し、３７℃、６時間培養した。細胞をＫＲＰ緩衝液（１４０ｍＭ ＮａＣｌ、２．７ｍＭ ＫＣｌ、０．９ｍＭ ＣａＣｌ₂ 、１．４７ｍＭＫＨ₂ＰＯ₄ 、８．０６ｍＭ Ｎａ₂ＨＰＯ₄ 、０．４９ｍＭ ＭｇＣｌ₂ 、０．１％ウシ血清アルブミン、ｐＨ７．４）で一回洗浄した後、０．５ｍｌの同緩衝液で３７℃、３０分間プレインキュベートした。その後２−デオキシ−Ｄ−［１−³Ｈ］グルコース（０．５μＣｉ／ウェル、１０μＭ）を加え、１０分間培養した。ウェルを２５ｍＭグルコースを含む氷冷ＰＢＳで３回洗浄して過剰の放射能を除いた後、０．１％ＳＤＳ処理して細胞破砕液を得、その放射能を液体シンチレーションカウンターにより測定した。なお、ヒトＴＭは、実施例に従い、ヒトＴＭのＥ１２３４５６ドメインを包含するペプチド（ＴＭＥ_1-6 )をチャイニーズハムスター卵母細胞（ＣＨＯ−Ｋ１細胞）で発現後、均一に精製したものを使用した。ＥＧＦはアップステートバイオテクノロジー社より購入して使用した。結果を表２に示す。
【００５４】
【表２】

表２から明らかなように、ＴＭＥ_1-6 は２−デオキシグルコース取り込みの促進作用を示した。また、ＵＴＭ０、ｒｓＴＭおよびＤ１２３も同様の作用を示した。
【００５５】
実施例４（製剤例１）
以下の成分を注射用蒸留水１０ｍｌに溶解し、無菌濾過したあとに１．０ｍｌずつ無菌バイアルに分注し、凍結乾燥して注射用製剤を調製した。
【００５６】
ＴＭＥ_1-6 ５ ｍｇ
精製ゼラチン ５０ ｍｇ
リン酸ナトリウム ３４．８ｍｇ
塩化ナトリウム ８１．８ｍｇ
マンニトール ２５ ｍｇ
【００５７】
実施例５（製剤例２）
以下の成分を注射用蒸留水１０ｍｌに溶解し、無菌濾過したあとに１．０ｍｌずつ無菌バイアルに分注し、凍結乾燥して注射用製剤を調製した。
【００５８】
ＵＴＭ０ ５ ｍｇ
アルブミン ２０ ｍｇ
リン酸ナトリウム ３４．８ｍｇ
塩化ナトリウム ８１．８ｍｇ
マンニトール ２５ ｍｇ
【００５９】
実施例６（製剤例３）
以下の成分を注射用蒸留水１０ｍｌに溶解し、無菌濾過したあとに１．０ｍｌずつ無菌バイアルに分注し、凍結乾燥して注射用製剤を調製した。
【００６０】
ｒｓＴＭ １０ ｍｇ
精製ゼラチン ５０ ｍｇ
リン酸ナトリウム ３４．８ｍｇ
塩化ナトリウム ８１．８ｍｇ
マンニトール ２５ ｍｇ
【００６１】
実施例７（製剤例４）
以下の成分を注射用蒸留水１０ｍｌに溶解し、無菌濾過したあとに１．０ｍｌずつ無菌バイアルに分注し、凍結乾燥して注射用製剤を調製した。
【００６２】
Ｄ１２３ １０ ｍｇ
アルブミン ２０ ｍｇ
リン酸ナトリウム ３４．８ｍｇ
塩化ナトリウム ８１．８ｍｇ
マンニトール ２５ ｍｇ
【００６３】
実施例８（製剤例５）
以下の成分を混合、撹拌して均一な組成物（軟膏剤）を調製した。
【００６４】
ＴＭＥ_1-6 ０．０１％
白色ワセリン ９６．８％
流動パラフィン ３ ％
ジブチルヒドロキシトルエン ０．２％
【００６５】
実施例９（製剤例６）
以下の成分を混合、撹拌して均一な組成物（軟膏剤）を調製した。
【００６６】
ＵＴＭ０ ０．０１％
ＩＧＦ−１ ０．０１％
白色ワセリン ９６．８％
流動パラフィン ３ ％
ジブチルヒドロキシトルエン ０．２％
【００６７】
【発明の効果】
本発明の創傷治癒剤ならびに細胞増殖および／または細胞成長促進剤は、組織損傷部位の細胞、例えば線維芽細胞などの細胞の成長あるいは増殖を促進する効果を発揮するヒトＴＭ様蛋白質、あるいはヒトＴＭ様蛋白質に加えてさらにヒト細胞成長因子を含有するので、種々の創傷、例えば、角膜潰瘍、放射線角膜傷、角膜移植、他の手術誘発眼内創傷のような眼創傷および切開、やけど、切り傷、破傷、深部術創、皮膚移植によるドナー部位創および潰瘍（皮膚、とこずれ、静脈うっ血および糖尿病）のような皮膚創のごとき上皮損傷、あるいは消化管の潰瘍、口腔・気道または尿路のごとき粘膜の損傷などの治療に用いることができる。さらに、乾せん、日焼け、皮膚発疹のような皮膚傷害にも用いることができる。
【００６８】
また、本発明の製剤は、上記の創傷に対し、公知の製剤に比較して、より優れた作用を有しており、創傷治癒剤として有用である。
【図面の簡単な説明】
【図１】図１は、ＴＭＥ_1-6 のスイス３Ｔ３細胞の増殖に及ぼす影響を示したグラフである。数値は、平均値±標準誤差で表している。
【図２】図２は、ＴＭＥ_1-6 のスイス３Ｔ３細胞のＤＮＡ合成に及ぼす影響を示したグラフである。
【図３】図３は、ＴＭＥ_1-6 、ｒｓＴＭおよびＵＴＭ０のスイス３Ｔ３細胞のＤＮＡ合成に及ぼす影響を示したグラフである。数値は、平均値±標準偏差で表している。[0001]
[Industrial application fields]
The present invention comprises a wound healing agent and a cell proliferation and / or cell growth promoter characterized by containing a thrombomodulin (hereinafter abbreviated as TM) -like protein, and a TM-like protein and human growth factor And a cell growth and / or cell growth promoter.
[0002]
[Prior art]
The wound healing mechanism is similar in skin, connective tissue and support tissue. The tissue here includes not only skin, bone, and cartilage but also support fascia made of a collagen matrix that wraps around muscles and organs.
[0003]
While the healing process at the wound site usually occurs through complex biological mechanisms involving several groups of specialized cells or proteins, it is well known that growth factors are essential. Growth factors are released into the wound area and migrate into the wound, where they stimulate keratinocyte and fibroblast proliferation or growth, initiate angiogenesis, and remodel the matrix and wound area [Tendijk et al: Bio / Technol. 7, 793-798, 1989].
[0004]
At this time, fibroblasts are also important as cells that synthesize and release new collagen. Other proteins produced by fibroblasts contribute to the substrate. One such protein, fibronectin, is an important substance that promotes the binding of major cells to the underlying substrate, and its synthesis is enhanced during wound repair.
[0005]
In this way, growth factors are released in a timely manner in the damaged part of the living body, and cell migration / proliferation / differentiation is promoted alone or jointly. Growth factors involved in wound healing include epidermal growth factor (hereinafter abbreviated as EGF), fibroblast growth factor (hereinafter abbreviated as FGF), insulin-like growth factor (hereinafter abbreviated as IGF), insulin, and platelet-derived Growth factors (hereinafter abbreviated as PDGF) are well known. EGF not only promotes proliferation or growth of fibroblasts, vascular endothelial cells, and epithelial cells, but also promotes epithelial cell migration, and plays a central role in epithelial regeneration in the tissue repair process. FGF not only promotes proliferation or growth of fibroblasts, vascular smooth muscle cells, and vascular endothelial cells, but also promotes migration of vascular endothelial cells and plays a central role in vascular regeneration. As IGF, it is known that there are two kinds of molecular species, IGF-1 and IGF-2. IGF acts in cooperation with PDGF to promote fibroblast proliferation or growth and normal differentiation and proliferation of epithelial cells. Currently, clinical application research is underway for each of these factors, but the effect as a wound healing agent is not necessarily sufficient, and there is a strong need for more effective growth factors or wound healing agents. It is.
[0006]
On the other hand, TM was discovered by Esmon et al. In 1981 as an anticoagulant existing on the surface of vascular endothelial cells [Procedures of the National Academy of Sciences of USA (Pro. Natl. Acad. Sci. USA.). 78, 2249-2254, 1981], and then the entire primary structure was clarified by Suzuki et al. In 1987 [EMBO J., 6, 1891-1897, 1987;Biochemistry (Biochem.2)6, 4350-4357, 1987] glycoprotein. TM binds to thrombin in a 1: 1 ratio and suppresses thrombin's procoagulation-promoting action and remarkably promotes protein C activation by thrombin, thereby effectively inhibiting the coagulation system. DIC), which has been suggested to be useful for the prevention and treatment of various thrombosis, etc. In recent years, various human TM-like proteins having a partial structure of the amino acid sequence of TM [specialized] Kaihei 1-6219,Biokimika Biophysica Actor (Biochim. Biophys. Acta.1)205, p162-170, 1994], clinical application as a DIC therapeutic agent is being attempted.
[0007]
Thus, TM is widely considered to be a molecule that acts on the coagulation and fibrinolysis system, but on the other hand, little is known about the effect on cell function.
[0008]
Imada et al. Have the same function as mouse TM when mouse tetracarcinoma F9 cells are differentiated into endoderm cells with retinoic acid or cyclic AMP (cAMP), and as a result of gene cloning, they are exactly the same as mouse TM. Fetomodulin (hereinafter abbreviated as FM), which has been revealed to be expressed in endoderm cells, and TM / FM not only in blood vessels but also in direct contact with tissue fluid, Alternatively, it has been reported that the interaction between cells, such as alveoli, neuroepithelium, atria, sinus nodules, and cardiac pacemakers during the middle to late stages of embryogenesis [developmental Biology (Dev. Biol.) 140, 113-122, 1990].
[0009]
Although the above reports are seen, there is no known report describing that TM has a direct proliferation and / or growth promoting action on cells.
[0010]
[Problems to be solved by the invention]
It is an object of the present invention to provide a more effective and safer wound healing agent and cell proliferation and / or cell growth promoter containing a new cell growth factor that can be an essential therapeutic agent for wounds as an active ingredient. It is a problem.
[0011]
[Means for Solving the Problems]
While the present inventor is conducting research on the proliferation or growth promoting effect of TM on various cells, the TM directly acts on Swiss 3T3 cells at a much lower concentration than the concentration at which TM exhibits anticoagulant action. In addition, the present inventors have found that it promotes proliferation and, further, finds the fact that it exhibits higher activity when used in combination with other growth factors.
[0012]
  When the first aspect of the present invention binds to thrombin and suppresses the coagulation promoting action of thrombin,
Both TM-like proteins have the activity of remarkably promoting the activation of thrombin protein C.
It is a wound healing agent characterized by containing white matter as an active ingredient. TM-like protein
Lower amino acid sequence1It is preferable to have.
(Amino acid sequence 1)
1 10
Gln Gly His Trp Ala Arg Glu Ala Pro Gly
11 20
Ala Trp Asp Cys Ser Val Glu Asn Gly Gly
21 30
Cys Glu His Ala Cys Asn Ala Ile Pro Gly
31 40
Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala
41 50
Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr
51 60
Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu
61 70
Cys Glu His Phe Cys Val Pro Asn Pro Asp
71 80
Gln Pro Gly Ser Tyr Ser Cys Met Cys Glu
81 90
Thr Gly Tyr Arg Leu Ala Ala Asp Gln His
91 100
Arg Cys Glu Asp Val Asp Asp Cys Ile Leu
101 110
Glu Pro Ser Pro Cys Pro Gln Arg Cys Val
111 120
Asn Thr Gln Gly Gly Phe Glu Cys His Cys
121 130
Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu
131 140
Cys Val Glu Pro Val Asp Pro Cys Phe Arg
141 150
Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu
151 160
Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala
161 170
Glu Gly Phe Ala Pro Ile Pro His Glu Pro
171 180
His Arg Cys Gln Met Phe Cys Asn Gln Thr
181 190
Ala Cys Pro Ala Asp Cys Asp Pro Asn Thr
191 200
Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr
201 210
Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp
211 220
Ile Asp Glu Cys Glu Asn Gly Gly Phe Cys
221 230
Ser Gly Val Cys His Asn Leu Pro Gly Thr
231 240
Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala
241 250
Leu Val Arg His Ile Gly Thr Asp Cys Asp
251 253
Ser Gly Lys
  However, the 242nd Val may be replaced with Ala.
[0015]
  A second aspect of the present invention is a wound healing agent comprising a TM-like protein and human cell growth factor. Here, as the TM-like protein, the amino acid sequence described above is the same as in the first aspect described above.1What has is preferable. The human cell growth factor is preferably human IGF-1, human insulin, human EGF or human basic FGF (hereinafter abbreviated as b-FGF).
[0016]
A combination of a TM-like protein and a human cell growth factor includes a combination of the above-described TM-like protein having amino acid sequence 1 and at least one of human IGF-1, human insulin, human EGF and human b-FGF, A combination of a TM-like protein having feature 1 and at least one of human IGF-1, human insulin, human EGF and human b-FGF, or a TM-like protein having feature 2 described above, human IGF-1, and human A combination with at least one of insulin, human EGF and human b-FGF is preferred.
[0017]
  A third aspect of the present invention is a cell proliferation and / or cell growth promoter characterized by containing a TM-like protein as an active ingredient. Preferably, it is a granulation tissue formation promoter. The TM-like protein used in the third aspect of the present invention is the same as the TM-like protein used in the first aspect of the present invention described above, that is, the following amino acid sequence:1What has is preferable.
[0018]

However, the 242nd Val may be replaced with Ala.
[0021]
  A fourth aspect of the present invention is a cell proliferation and / or cell growth promoter characterized by containing a TM-like protein and human cell growth factor. Preferably, it is a granulation tissue formation promoter. Here, as the TM-like protein, the above-mentioned amino acid sequence is the same as in the above-mentioned third aspect.1What has is preferable. The human cell growth factor is preferably human IGF-1, human insulin, human EGF or human b-FGF.
[0022]
  As a combination of a TM-like protein and a human cell growth factor, a combination of the above-described TM-like protein having amino acid sequence 1 and at least one of human IGF-1, human insulin, human EGF and human b-FGFSegapreferable.
[0023]
Hereinafter, the present invention will be described in detail.
[0024]
  As used herein, the term “TM-like protein” refers to TM and TM biological or immunological activity (In other words, it binds to thrombin and suppresses thrombin's procoagulant action and significantly promotes the activation of thrombin protein C)It means a peptide having a partial amino acid sequence necessary for the above or an analog thereof.In this case, the term “peptide” means, as will be described later, a peptide showing the biological or immunological activity of TM and TM in the full-length 557 amino acid residues constituting human TM. Means a fragment. The term “analogue thereof” means a product obtained by modifying or deleting a sugar chain such as an O-glycoside-linked sugar chain or an N-glycoside-linked sugar chain of the aforementioned peptide fragment.The term “wound” is a general term for tissue damage caused by external force, but it creates a releasable injury in which the continuity of the skin and mucous membrane surface is cut off, and a closed injury that maintains continuity as a wound. “Wound healing” is an exudation in which an inflammatory reaction occurs, hyperpermeability of capillaries and the like, migration of leukocytes, etc. occurs, and bacteria and necrotic substances are removed by phagocytosis and fibrin deposition occurs. The rupture phase, the formation of capillaries and the proliferation of fibroblasts, the appearance of granulation, the fibrin being absorbed and replaced by collagen, the growth phase of the fibrin and the new blood vessels and exudate cells begin to decrease, and the amount of collagen increases. It is said that it takes a turning point in the maturity when wound contraction occurs [Nanzandou Medical Dictionary, Nanzando, pages 1156 to 1157, 1990], and these meanings are also included in the present invention. Granulation (tissue) is a young connective tissue that is actively proliferating, and is an important tissue formed during wound healing that absorbs the necrotic tissue of the wound, fills the defect, and causes fibrosis. Fibroblasts are the basic constituent cells of granulation tissue. The term “wound healing agent” also refers to various wounds such as corneal ulcers, radiation corneal wounds, corneal transplants and other surgically induced intraocular wounds, and incisions, burns, cuts, injuries, deep Surgery wounds, donor site wounds by skin grafts and epithelial damage such as skin wounds such as ulcers (skin, mess, venous congestion and diabetes), or digestive tract ulcers, damage to mucous membranes such as oral cavity, airway or urinary tract, etc. Furthermore, it broadly encompasses therapeutic agents for promoting healing of skin disorders such as psoriasis, sunburn, and skin rash.
[0025]
The TM-like protein used in the present invention may be a natural type or a gene-engineered protein, and may be a modified type or a chimeric type obtained by a genetic engineering technique. In the case of a pharmaceutical product, preferably a human-derived TM is desired. In addition, human-derived TM is membrane-bound, and those having a full-length amino acid sequence have low solubility in water. For this reason, in the present invention, TM having a full-length amino acid sequence and high solubility (hereinafter abbreviated as soluble TM) is more preferable.
[0026]
Such TM is, for example, TM derived from human placenta [Japanese Patent Laid-Open No. 60-199819]. Soluble TM includes human urine-derived soluble TM having a molecular weight of 55,000 to 58,000 and 60,000 to 65,000 in a non-reduced state [JP-A-3-86900] and a molecular weight in a non-reduced state. Examples include 72,000 ± 3,000 and 79,000 ± 3,000 human urine-derived soluble TM [JP-A-3-218399]. Of these, human urine-derived soluble TM having molecular weights of 72,000 ± 3,000 and 79,000 ± 3,000 is preferable. These human urine-derived soluble TMs have the feature 1 or feature 2 of the TM-like protein that can be preferably used in the present invention, as described in JP-A-3-218399. .
[0027]
Moreover, the TM-like protein used in the present invention can be produced by a gene recombination technique. For use in human therapeutic agents, those having human amino acid sequences are preferred. Specifically, the primary structure of human TM produced by genetic engineering techniques and shown by Suzuki et al.Emboss Journal (EMBO. J. et al.)6, 1891-1897, 1987;Biochemistry (Biochem.)26, 4350-4357, 1987] and fragments consisting of a partial amino acid sequence necessary for biological or immunological activity thereof.
[0028]
According to Suzuki et al., The full-length human TM is composed of 557 amino acid residues, which are composed of five domain structures, D1, D2, D3, D4 and D5, in order from the N-terminus. Moreover, it has been reported that the anticoagulant activity is limited to the 4th to 6th domains (E456) of the D2 domain consisting of repeating 6 epidermal growth factor (EGF) -like structures. Examples of fragments that can be used in the healing agent of the present invention include various peptide fragments including the E456 peptide structure disclosed by Suzuki et al. [Japanese Patent Laid-Open No. 1-6219] or soluble TM-like proteins disclosed by Nawa et al. Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.) 171, 729-737, 1990]. More specifically, it is composed of amino acid residues 214 to 466 from the N terminus of human TM, including the first to sixth domain structures of the EGF-like structure of the D2 domain (hereinafter referred to as E123456). Peptide (hereinafter TME)_1-6), A peptide composed of the 1-491th amino acid residue (hereinafter referred to as rsTM), a peptide composed of the 227-462th amino acid residue (D2), and the 1-497th amino acid residue A peptide composed of the amino acid residues 1-462 (D12), a peptide composed of the amino acid residues 227-497 (D23), 1-557 Peptide (D12345) composed of the amino acid residue of the th (D12345), peptide (E456) composed of the amino acid residues of 345 to 462, etc. are preferred. Among these fragments, the fragment composed of amino acid residues 214 to 466 (TME) including the second domain of TM, that is, the first to sixth structures of the EGF-like structural domain._1-6), That is, those having the amino acid sequence 1 described above, rsTM or D123 are most preferred.
[0029]
In addition, the above-mentioned fragment is a modified product obtained by deleting a part of TM or the above peptide structure using a gene recombination technique, or substituting or modifying an amino acid with another amino acid, and an O-glycoside-linked sugar chain or N-glycoside. An analog obtained by modifying or deleting a sugar chain such as a binding sugar chain may also be used. Specifically, some of the constituent amino acids of the above polypeptide lacks [WO92 / 00325, JP-A-2-255699, EP0474273A2], or those substituted with other amino acids [WO91 / 15514, WO93 / 155755,JPNo. 5-310787]. In addition, those in which oxidation is prevented by substituting methionine with other amino acids [WO91 / 15514], or those in which degradation by proteolytic enzymes is prevented by modifying the amino acid sequence [WO93 / 15755] can be mentioned. In addition, chondroitin and / or a chondroitin sulfate sugar chain containing an acidic amino acid sequence derived from bovine thrombomodulin having a sugar chain containing chondroitin sulfate and / or chondroitin sulfate [WO91 / 04276, WO91 / 05803, EP0412841A1] Containing chain [EP0488317A2], modified O-glycosylation site sugar chain or deleted O-glycosylation site region [WO92 / 03149], including chondroitin and / or chondroitin sulfate Nothing [JP-A-4-210700] and the like.
[0030]
The second aspect and the fourth aspect of the present invention contain human cell growth factor as an active ingredient in addition to the TM-like protein as described above. Thereby, it is possible to obtain a large healing effect and a promoting effect when the wound healing agent and the cell proliferation and / or cell growth promoting agent are composed of TM-like protein or human cell growth factor alone.
[0031]
In the present invention, as the human cell growth factor, it is preferable to use one kind selected from human IGF-1, human insulin, human EGF, and human b-FGF, or a combination of two or more kinds.
[0032]
In the present invention, human IGF-1, human insulin, human EGF, and human b-FGF can be contained as cell growth factors as described above, but are not limited thereto. Any substance having IGF, insulin, EGF, or FGF-like activity may be used.[Protein Nucleic Acid Enzyme, 36, 7, 1078-1087, 1237-1246, 1991, Clinical Immunity, 27,Supplement (Suppl.)16, pp. 307-316, 1995]. For example, naturally-occurring substances produced in the body of animals and animal cells, substances produced by gene recombination technology, and gene recombination techniques can be used to delete some of these structures or to replace amino acids with other amino acids. Substituted or modified variants and analogs with modified or deleted sugar chains are exemplified. Preferably, those derived from human or those having a structure equivalent to that derived from human are preferred. Some of these are available as commercial products. Moreover, as such a human cell growth factor, what was obtained by any well-known pharmaceutical manufacturing method can be used.
[0033]
Any of the healing agents of the first and second aspects of the present invention and any of the cell proliferation and / or cell growth promoting agents of the third and fourth aspects of the present invention are made into various dosage forms. In this case, various additives and other components can be blended. For example, TM-like protein can be used as a suitable carrier or medium generally used as a drug, such as sterilized water or physiological saline, vegetable oil (eg, sesame oil, olive oil), mineral oil (eg, petroleum jelly, paraffin), higher alcohol ( Examples, cetanol, stearyl alcohol), higher fatty acids (eg, stearic acid, palmitic acid), harmless organic solvents (eg, ethanol), etc., and further excipients (eg, mannitol, carboxymethyl cellulose) as necessary Agent, emulsifier (eg, cholesterol), suspending agent (eg, gum arabic), surfactant (eg, polyoxyethylene hydrogenated castor oil surfactant, polyethylene glycol surfactant), solubilizer (eg, Surfactant, sodium phosphate, sodium acetate), adsorption inhibitor (eg, nonionic surfactant, gelatin), stable In combination with agents (eg, amino acids, sugars, sugar alcohols, albumin) or preservatives (eg, benzyl alcohol, parabens), moisturizers (eg, urea, cholesterol), antioxidants (eg, tocopherols), etc. It can be prepared into pharmaceutical preparations such as injections, transdermal absorption agents, eye drops, nasal absorption agents, oral preparations, etc., preferably injections or transdermal absorption agents, which are suitable for effective administration. For example, as a preparation for injection, it can be provided in a lyophilized product, a liquid for injection, or a form enclosed in an osmotic pump. Moreover, as a transdermal absorption agent, it can provide with a liquid agent, an ointment, a cream agent, a gel agent, a poultice, artificial skin, etc.
[0034]
Wound healing agent of the present inventionAnd cell proliferation and / or cell growth promoterAs a method of administration, for example, the amount of TM-like protein is about 0.01 to 1000 μg / kg per day, more preferably 0.1 to 1000 μg / kg by a method such as intravenous injection, subcutaneous injection, intramuscular injection or oral administration. It can be administered continuously by administering an amount of 100 μg / kg or enclosing it in an osmotic pump or the like and placing it in a living body. A preparation containing human IGF-1, human insulin, human EGF, or human b-FGF as an active ingredient in addition to the TM-like protein, each factor is administered at the same time as the above TM-like protein administration. It is preferable to administer an amount of 0.01 to 1000 μg / kg, more preferably 0.1 to 100 μg / kg.
[0035]
In addition, as a transdermal absorbent, by applying locally to the burn or wound site by a method such as liquid, ointment, cream, gel, buccal, etc., for example, the TM-like protein concentration is about 0.001 to 1000 μg. / Ml, more preferably at a concentration of 0.01-100 μg / ml. In addition to the human TM-like protein, a preparation containing human IGF-1, human insulin, human EGF or human b-FGF as an active ingredient, each factor is added to the above-mentioned TM-like protein at the same time each day. An amount of 0.01 to 1000 μg / ml, more preferably 0.1 to 100 μg / ml is applied.
[0036]
Further, in the case of cultured skin transplantation used in the case of burns, for example, the concentration of human TM-like protein is about 0.001 to 1000 μg / ml, more preferably 0.01 to 100 μg / ml in the cell culture medium. Can also be added. In addition to TM-like protein, human IGF-1, human insulin, human EGF or human b-FGF was added simultaneously with TM-like protein, and each factor was about 0.01 to 1000 μg / ml, more preferably 0 It can also be added at a concentration of 1 to 100 μg / ml.
[0037]
[Action]
Since the wound healing agent of the present invention contains, as an active ingredient, a TM-like protein that exerts a growth and proliferation promoting effect on cells such as fibroblasts, or both TM-like protein and human cell growth factor, Ocular wounds and incisions such as, for example, corneal ulcers, radiation corneal wounds, keratoplasty and other surgically induced intraocular wounds, burns, cuts, injuries, deep surgical wounds, donor site wounds and ulcers by skin transplantation (skin, misplaced) It can be used for the treatment of epithelial damage such as skin wounds such as venous congestion and diabetes, or mucosal damage such as ulcers of the digestive tract, oral cavity, respiratory tract or urinary tract. Furthermore, it can be used for skin injuries such as psoriasis, sunburn, and skin rash.
[0038]
The cell proliferation and / or cell growth promoter of the present invention particularly promotes the proliferation / growth of fibroblasts and acts to promote granulation tissue formation. In addition, cell proliferation can be promoted by adding to the cell culture system. The human cells can also be cultured by adding to a culture solution of human cells used for transplantation.
[0039]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples of obtaining TM-like proteins and examples of the present invention. However, TM-like proteins used in the present invention are limited to those described in the following examples. In addition, the present invention is not limited to the following examples.
[0040]
Acquisition Example 1 (Example of human TM-like protein acquisition: TME_1-6 )
PRS7TM was prepared according to the method of Nawa et al. [Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.) 171, 729-737, 1990]. Specifically, a TM gene fragment obtained from a human cDNA library was inserted into a pUC119 expression vector, followed by restriction enzyme treatment to prepare pRS7TM. Furthermore, the TM signal sequence of pRS7TM was replaced with the interferon γ signal sequence, and the N-terminal sequence was deleted. That is, a fragment containing a new signal sequence of 80 bp was obtained by treatment with Sal-BstN1 restriction enzyme, a fragment encoding the first EGF domain was obtained by treatment with PstI restriction enzyme, and then linked with a linker to prepare pTMP22. This pTMP22 was treated with HindIII-MluI restriction enzyme, and the resulting fragment was replaced with the HindIII-MluI fragment of pTMs07 to prepare pP7TM. In order to delete the Ser / Thr rich region, pTMs07 was treated with HincII restriction enzyme to isolate the fragment, and further treated with HpaII restriction enzyme to obtain a 70 bp fragment encoding the sixth EGF domain. . A linker containing a stop codon was ligated after Lys-466 of this fragment, followed by Apa I-Xba I restriction enzyme treatment, and the resulting fragment was incorporated into the Apa I-Xba I site of pP7TM to prepare pPG3TM.
[0041]
This plasmid (pPG3TM) was transfected into a Chinese hamster oocyte line (CHO-K1 cell line) according to a conventional method. These cells were further cultured in a selective medium for 2 to 3 weeks to select stable transformed cells, and the resulting colonies were further cultured. The culture supernatant was purified by anti-TM monoclonal antibody column and gel filtration, and TME_1-6 Got.
[0042]
Acquisition example 2 (soluble TM acquisition example: UTM0)
It was prepared according to the method described in JP-A-3-218399. Ie
Then, 100 L of raw urine was filtered with acrylic fiber to adsorb and remove urokinase in urine, and the passing urine was desalted and concentrated using an ultrafiltration membrane. Subsequently, purification was performed sequentially using DEAE cellulose (manufactured by Whatman) column, DIP-thrombin-agarose chromatography and Sephacryl S-300 (manufactured by Pharmacia Fine Chemical), and the active fraction was collected (hereinafter referred to as UTM0). . This fraction was dialyzed overnight against distilled water and lyophilized.
[0043]
Acquisition Example 3 (Soluble TM acquisition example: rsTM)
PRS7TM was prepared according to the method of Nawa et al. [Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.) 171, 729-737, 1990]. That is, a TM gene fragment obtained from a human cDNA library was inserted into a pUC119 expression vector, followed by restriction enzyme treatment and the like to prepare plasmid pTMs07 encoding rsTM. In addition, Neo^r The gene was integrated to prepare an expression plasmid pTMs07-neo.
[0044]
This plasmid (pTMs07-neo) was transfected into a Chinese hamster oocyte line (CHO-K1 cell line) according to a conventional method. These cells were further cultured in a selective medium for 2 to 3 weeks to select stable transformed cells, and the resulting colonies were further cultured. The culture supernatant was purified by anti-TM monoclonal antibody column and gel filtration to obtain rsTM.
[0045]
Acquisition example 4 (soluble TM acquisition example: D123)
It manufactured according to the method of WO92 / 00325 gazette. That is, by using DNA picked up from a human placenta cDNA library, a vector expressing a soluble thrombomodulin consisting of amino acid 497 residues having an amino-terminal amino acid sequence of Ala-Pro-Ala- was prepared, and this was expressed in CHO cells. After integration, gene amplification was performed to obtain a high expression strain. The culture solution of this highly expressing strain was purified by DIP-thrombin-agarose column and gel filtration to obtain D123.
[0046]
Example 1 (Effect of human TM on fibroblasts 1: growth promoting effect)
Swiss 3T3 cells (Japan Cancer Research Bank) are suspended in DMEM medium (Gibco) containing 10% fetal bovine serum (hereinafter abbreviated as CS: Gibco) and 1 × 10 per well in a 35 mm culture plate. ⁴ Seeded at an individual density of 37 ° C., 5% CO₂/ 95% Air. After 24 hours, the medium was changed to DMEM medium (0.1 to 10% CS) containing various concentrations of CS containing 50 ng / ml of human TM, and thereafter the medium was changed once every two days (see arrow in FIG. 1). . The cells were treated with 0.25% trypsin / 0.02% EDTA and the cell number was measured. According to the method described in Acquisition Example 1, human TM is obtained by expressing a peptide (TME1-6) containing human TM E123456 in Chinese hamster oocytes (CHO-K1 cells) and then purifying it uniformly. used.
[0047]
The result is shown in FIG. As is clear from FIG. 1, TME_1-6 Showed cell proliferation effect in the presence of 0.1% or 1% serum. UTM0, rsTM and D123 also exhibit the same action.
[0048]
In general, the ability to promote proliferation in vitro is directly related to cell differentiation that can cause tissue growth in vivo, and a substance having a strong ability to promote proliferation in vitro also has a growth stimulating effect in vivo. Is well known in the study of growth factors. Therefore, it can be seen that the human TM-like protein has an effect even in a living body.
[0049]
Example 2 (Effect of human TM on fibroblasts 2: DNA synthesis promoting effect)
Swiss 3T3 cells are suspended in DMEM medium containing 10% CS, seeded in a 96-well microtiter plate, 37 ° C., 5% CO 2.₂ / 95% Air condition until confluent. Subsequently, the culture medium was replaced with a DMEM medium containing (or not containing) 0.1% CS, and cultured for 48 hours, whereby the cells were completely inactive. Next, as shown in Table 1 and FIG. 2, various concentrations of human TM and various concentrations of growth factors were added and cultured at 37 ° C. for 20 hours. afterwards^ThreeH-thymidine (1 μCi / well) was added and further cultured for 8 hours. The wells were washed with ice-cold phosphate buffer (hereinafter abbreviated as PBS).^ThreeAfter removing H-thymidine, the cells were detached from the plate by treatment with 0.25% trypsin / 0.02% EDTA and collected on a glass fiber filter. After washing the cells with ice-cooled trichloroacetic acid, the radioactivity remaining on the filter was measured with a liquid scintillation counter. In addition, human TM is a peptide (TME) containing E123456 of human TM according to Acquisition Example 1._1-6 ) Was expressed in Chinese hamster oocytes (CHO-K1 cells) and then purified uniformly, human urine-derived soluble TM (UTM0) purified according to Acquisition Example 2 and rsTM purified according to Acquisition Example 3 were used. IGF-1 and bFGF were purchased from Intergen, insulin was purchased from Wako Pure Chemicals, and EGF was purchased from Upstate Biotechnology.
[0050]
The results are shown in FIGS. 2 and 3 and Table 1.
[0051]
As is clear from FIG. 2, TME_1-6 Showed a DNA synthesis promoting action in the presence or absence of serum. As is clear from FIG. 3, TME_1-6, RsTM and UTM0 both showed DNA synthesis promoting action, and UTM0 showed the strongest action.
[0052]
[Table 1]

As is clear from Table 1, TME_1-6 Not only shows DNA synthesis promoting action alone, but also shows additive action with EGF and b-FGF known as cell growth factors, and synergistic DNA synthesis promoting action with insulin or IGF-1. showed that.
UTM0, rsTM and D123 also showed the same action.
[0053]
Example 3 (Effect of fibroblasts by human TM 3: glucose transport promoting effect)
This was carried out according to the method of Hamana et al. [Experimental Cell Research (Exp. Cell Res.) 186, 83-89, 1990]. Specifically, Swiss 3T3 cells were suspended in DMEM medium containing 10% CS, seeded on a 24-well multiplate, and incubated at 37 ° C., 5% CO 2.₂ / 95% Air condition until confluent. Next, the culture solution was replaced with a DMEM medium containing 0.5% CS and cultured for 48 hours, and then 50 ng / ml human TM, 10 ng / ml EGF or 10% CS was added, and the cells were cultured at 37 ° C. for 6 hours. Cells were washed with KRP buffer (140 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl₂ 1.47 mM KH₂PO_Four 8.06 mM Na₂HPO_Four 0.49 mM MgCl₂ , 0.1% bovine serum albumin, pH 7.4), and then preincubated with 0.5 ml of the same buffer at 37 ° C. for 30 minutes. Then 2-deoxy-D- [1-^ThreeH] glucose (0.5 μCi / well, 10 μM) was added and incubated for 10 minutes. The wells were washed 3 times with ice-cold PBS containing 25 mM glucose to remove excess radioactivity, and then treated with 0.1% SDS to obtain a cell lysate, and the radioactivity was measured with a liquid scintillation counter. In addition, human TM is a peptide containing the E123456 domain of human TM (TME) according to the Examples._1-6 ) Was expressed in Chinese hamster oocytes (CHO-K1 cells) and then purified to homogeneity. EGF was purchased from Upstate Biotechnology and used. The results are shown in Table 2.
[0054]
[Table 2]

As is clear from Table 2, TME_1-6 Showed the promoting action of 2-deoxyglucose uptake. UTM0, rsTM and D123 also showed the same action.
[0055]
Example 4 (Formulation Example 1)
The following components were dissolved in 10 ml of distilled water for injection, filtered aseptically, dispensed 1.0 ml each into a sterile vial, and lyophilized to prepare an injectable preparation.
[0056]
TME_1-6               5 mg
Purified gelatin 50 mg
Sodium phosphate 34.8mg
Sodium chloride 81.8mg
Mannitol 25 mg
[0057]
Example 5 (Formulation Example 2)
The following components were dissolved in 10 ml of distilled water for injection, filtered aseptically, dispensed 1.0 ml each into a sterile vial, and lyophilized to prepare an injectable preparation.
[0058]
UTM0 5 mg
Albumin 20 mg
Sodium phosphate 34.8mg
Sodium chloride 81.8mg
Mannitol 25 mg
[0059]
Example 6 (Formulation Example 3)
The following components were dissolved in 10 ml of distilled water for injection, filtered aseptically, dispensed 1.0 ml each into a sterile vial, and lyophilized to prepare an injectable preparation.
[0060]
rsTM 10 mg
Purified gelatin 50 mg
Sodium phosphate 34.8mg
Sodium chloride 81.8mg
Mannitol 25 mg
[0061]
Example 7 (Formulation Example 4)
The following components were dissolved in 10 ml of distilled water for injection, filtered aseptically, dispensed 1.0 ml each into a sterile vial, and lyophilized to prepare an injectable preparation.
[0062]
D123 10 mg
Albumin 20 mg
Sodium phosphate 34.8mg
Sodium chloride 81.8mg
Mannitol 25 mg
[0063]
Example 8 (Formulation Example 5)
The following components were mixed and stirred to prepare a uniform composition (ointment).
[0064]
TME_1-6                   0.01%
White petrolatum 96.8%
Liquid paraffin 3%
Dibutylhydroxytoluene 0.2%
[0065]
Example 9 (Formulation Example 6)
The following components were mixed and stirred to prepare a uniform composition (ointment).
[0066]
UTM0 0.01%
IGF-1 0.01%
White petrolatum 96.8%
Liquid paraffin 3%
Dibutylhydroxytoluene 0.2%
[0067]
【The invention's effect】
The wound healing agent and cell proliferation and / or cell growth promoter of the present invention are human TM-like proteins that exhibit the effect of promoting the growth or proliferation of cells at the site of tissue damage, such as fibroblasts, or human TM In addition to protein-like protein, it contains human cell growth factor, so various wounds such as corneal ulcers, radiation corneal wounds, corneal transplants, eye wounds and incisions such as other surgically induced intraocular wounds, burns, cuts, Epithelial damage such as rupture, deep surgery wound, donor site wound and skin ulcer (skin, misplacement, venous congestion and diabetes), or mucosa such as gastrointestinal ulcer, oral / respiratory tract or urinary tract It can be used for the treatment of injuries. Furthermore, it can be used for skin injuries such as psoriasis, sunburn, and skin rash.
[0068]
In addition, the preparation of the present invention has a more excellent effect on the above wound than the known preparations and is useful as a wound healing agent.
[Brief description of the drawings]
FIG. 1 shows TME_1-6 It is the graph which showed the influence which it has on the proliferation of Swiss 3T3 cell. Numerical values are expressed as mean ± standard error.
FIG. 2 shows TME_1-6 It is the graph which showed the influence which acts on the DNA synthesis | combination of Swiss 3T3 cell.
FIG. 3 shows TME_1-6 , RsTM and UTM0 are graphs showing the influence of Swiss 3T3 cells on DNA synthesis. The numerical value is expressed as an average value ± standard deviation.

Claims (8)

Bound to thrombin, thrombomodulin-like protein having an activity of significantly promoting the protein C activation of thrombin suppresses the procoagulant action of thrombin containing as an active ingredient, the thrombomodulin-like protein, the N-terminus of human thrombomodulin A wound healing agent characterized by being a peptide comprising the amino acid residues at positions 345 to 462, or a variant obtained by deleting a part of the structure, or a variant obtained by substituting or modifying an amino acid with another amino acid . The wound healing agent according to claim 1, wherein the thrombomodulin-like protein has the following amino acid sequence 1.
(Amino acid sequence 1)
1 10
Gln Gly His Trp Ala Arg Glu Ala Pro Gly
11 20
Ala Trp Asp Cys Ser Val Glu Asn Gly Gly
21 30
Cys Glu His Ala Cys Asn Ala Ile Pro Gly
31 40
Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala
41 50
Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr
51 60
Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu
61 70
Cys Glu His Phe Cys Val Pro Asn Pro Asp
71 80
Gln Pro Gly Ser Tyr Ser Cys Met Cys Glu
81 90
Thr Gly Tyr Arg Leu Ala Ala Asp Gln His
91 100
Arg Cys Glu Asp Val Asp Asp Cys Ile Leu
101 110
Glu Pro Ser Pro Cys Pro Gln Arg Cys Val
111 120
Asn Thr Gln Gly Gly Phe Glu Cys His Cys
121 130
Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu
131 140
Cys Val Glu Pro Val Asp Pro Cys Phe Arg
141 150
Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu
151 160
Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala
161 170
Glu Gly Phe Ala Pro Ile Pro His Glu Pro
171 180
His Arg Cys Gln Met Phe Cys Asn Gln Thr
181 190
Ala Cys Pro Ala Asp Cys Asp Pro Asn Thr
191 200
Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr
201 210
Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp
211 220
Ile Asp Glu Cys Glu Asn Gly Gly Phe Cys
221 230
Ser Gly Val Cys His Asn Leu Pro Gly Thr
231 240
Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala
241 250
Leu Val Arg His Ile Gly Thr Asp Cys Asp
251 253
Ser Gly Lys The thrombomodulin-like protein is a peptide comprising the amino acid residues 1-497 from the N-terminal among peptides comprising the amino acid residues 345-462 from the N-terminal of human thrombomodulin. wound Osamu 癒剤. The wound according to any one of claims 1 to 3 , comprising at least one of human insulin-like cell growth factor-1, human insulin, human epithelial cell growth factor and basic fibroblast growth factor as the human cell growth factor. Healing agent. Bound to thrombin, thrombomodulin-like protein having an activity of significantly promoting the protein C activation of thrombin suppresses the procoagulant action of thrombin containing as an active ingredient, the thrombomodulin-like protein, the N-terminus of human thrombomodulin A cell growth and / or a peptide comprising the amino acid residues 345 to 462, or a variant in which a part of its structure is deleted or a variant obtained by substituting or modifying an amino acid with another amino acid Or cell growth promoter. The cell proliferation and / or cell growth promoter according to claim 5 , wherein the thrombomodulin-like protein has the following amino acid sequence 1.
(Amino acid sequence 1)
1 10
Gln Gly His Trp Ala Arg Glu Ala Pro Gly
11 20
Ala Trp Asp Cys Ser Val Glu Asn Gly Gly
21 30
Cys Glu His Ala Cys Asn Ala Ile Pro Gly
31 40
Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala
41 50
Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr
51 60
Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu
61 70
Cys Glu His Phe Cys Val Pro Asn Pro Asp
71 80
Gln Pro Gly Ser Tyr Ser Cys Met Cys Glu
81 90
Thr Gly Tyr Arg Leu Ala Ala Asp Gln His
91 100
Arg Cys Glu Asp Val Asp Asp Cys Ile Leu
101 110
Glu Pro Ser Pro Cys Pro Gln Arg Cys Val
111 120
Asn Thr Gln Gly Gly Phe Glu Cys His Cys
121 130
Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu
131 140
Cys Val Glu Pro Val Asp Pro Cys Phe Arg
141 150
Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu
151 160
Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala
161 170
Glu Gly Phe Ala Pro Ile Pro His Glu Pro
171 180
His Arg Cys Gln Met Phe Cys Asn Gln Thr
181 190
Ala Cys Pro Ala Asp Cys Asp Pro Asn Thr
191 200
Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr
201 210
Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp
211 220
Ile Asp Glu Cys Glu Asn Gly Gly Phe Cys
221 230
Ser Gly Val Cys His Asn Leu Pro Gly Thr
231 240
Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala
241 250
Leu Val Arg His Ile Gly Thr Asp Cys Asp
251 253
Ser Gly Lys 6. The thrombomodulin-like protein is a peptide including the amino acid residues 1-497 from the N-terminal among peptides including the amino acid residues 345-462 from the N-terminal of human thrombomodulin. Cell proliferation and / or cell growth promoter. The cell according to any one of claims 5 to 7 , comprising at least one of human insulin-like cell growth factor-1, human insulin, human epithelial cell growth factor and basic fibroblast growth factor as the human cell growth factor. Proliferation and / or cell growth promoter.

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