JP2004002412A - Therapeutic agent of nerve damage - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic agent of neurological function insufficient disease, such as central nervous system injury and cerebral infarctions including injury of spinal cord, having an excellent neurotization-promoting action, which is used in various administration methods including not only to pour into damaged section but also to inject intravenously, easily and conveniently handled and preserved for long period of time, and produced at any time and in large quantities. <P>SOLUTION: There is provided a therapeutic agent of neurological function insufficient disease, such as central nervous system injury and cerebral infarctions including injury of spinal cord, wherein the therapeutic agent contains as an effective component: one or more of substances selected from the group consisting of substances secreted from Dendritic Cell such as IL-12 , GM-CSF or the like, substances which induce / proliferate Dendritic Cell, substances which activate Dendritic Cell, substances which induce developing of neurotrophic factor in nervous tissue, substances which induce / proliferate micro glia or macrophage in nervous tissue, and a vector which develops the substances; or Dendritic Cell subset which secretes neurotrophic factor NT-3, CNTF, TGF-β1, IL-6, or EGF. <P>COPYRIGHT: (C)2004,JPO

Description

【００２８】
未成熟樹状細胞では、インビボでの神経再生効果やマイクログリアの増殖、食作用の増強を誘導するＮＴ−３、脊髄運動知覚両神経に対し変性・細胞死保護の効果を有するＣＮＴＦ、マイクログリアやマクロファージ由来の細胞障害性物質の放出の抑制作用を有するＴＧＦ−β１、各種ニューロン（コリン・カテコールアミン・ドーパミン作動性）に対する保護効果を誘導するＩＬ−６の発現が確認された（図７）。また、成熟樹状細胞では、ＮＴ−３、ＣＮＴＦ、ＴＧＦ−β１、ＩＬ−６に加え、神経保護効果の認められているＥＧＦの発現が確認された（図８）。それぞれの遺伝子について、ゲルからｃＤＮＡを抽出して塩基配列を解析し、発現産物がそれぞれＮＴ−３、ＣＮＴＦ、ＴＧＦ−β１、ＩＬ−６及びＥＧＦであることを確認した。
【００２９】
実施例８（神経栄養因子ＮＴ−３の分泌）
さらに神経再生のために最も重要と考えられている神経栄養因子の一つであるＮＴ−３に関して、実際に樹状細胞から分泌されているかをＮＴ−３イムノアッセイシステム（Ｐｒｏｍｅｇａ社製）を用いたＥＬＩＳＡ法にて解析した。実施例１と同様にして、生後６週齢のＢＡＬＢ／ｃ雌成熟マウスの脾臓から、免疫磁気ビーズ法によりＣＤ１１ｃ陽性の未成熟樹状細胞分離した。このＣＤ１１ｃ陽性の未成熟樹状細胞１×１０^５個を、ＲＰＭＩ１６４０＋１０％胎仔血清の培養液中で３７℃、５％ＣＯ_２下２４時間インキュベートした後、その培養上清を回収した。コントロールとしてＲＰＭＩ１６４０のみ、及びＣＤ４陽性Ｔ細胞、ＣＤ８陽性Ｔ細胞それぞれ１×１０^５個を用いた。２種の抗ＮＴ−３抗体を用いたサンドイッチＥＬＩＳＡ法により上清中のＮＴ−３を定量的に解析した結果、１×１０^５個の樹状細胞が２４時間で約１．７５ｎｇのＮＴ−３を分泌していることが明らかになった。ＲＰＭＩ１６４０のみ、及びＣＤ４陽性Ｔ細胞、ＣＤ８陽性Ｔ細胞では分泌は認められなかった（図９）。
【００３０】
実施例９（脊髄損傷に対する樹状細胞の効果の再確認）
生後６週齡のＢＡＬＢ／ｃ雌マウスをエーテル麻酔下に第８胸椎椎弓切除を行い、顕微鏡下で左側脊髄を半切した脊髄損傷モデルマウスを作製した。脊髄損傷部位に直ちに樹状細胞１×１０^６個を移植した後（ＤＣ，ｎ＝１７）、本発明者らが開発した下肢の運動機能評価法（行動解析装置ＳＣＡＮＥＴ　ＭＶ−１０を用いて、立ち上がり回数を自動的に解析する、ＲＧスコア）、及びすでに確立された下肢の運動機能評価法の一つであるＢＢＢスケール（０−２１点の間で評価し、０点は全く下肢運動が認められない、２１点は正常）を用いて、経時的に評価を行った。コントロールとしてＲＰＭＩ１６４０（ＲＰＭＩ，ｎ＝１８）、及びＣＤ８陽性Ｔ細胞（Ｔ，ｎ＝１０）を同様に損傷脊髄部位に移植した。結果を図１０に示す。図１０からもわかるように、２つの評価法（ＲＧスコア及びＢＢＢスケール）で共に、コントロールのＴ細胞やＲＰＭＩの場合と比較して、ＤＣ移植群で統計学的有意に高いスコアを示した。従って、神経栄養因子ＮＴ−３を分泌する樹状細胞サブセットを脊髄損傷部位に移植することにより、脊髄機能の回復が促進されることが再確認することができた。
【００３１】
実施例１０（樹状細胞移植による内在性マイクログリアの活性化）
樹状細胞移植により内在性マイクログリアや、損傷部の血管から侵入したマクロファージの反応性に変化がみられるかどうかを検討するため、それらを認識するＭａｃ−１抗体を用いて、免疫組織染色を行い、陽性細胞数の経時的な変化を調べた。まず、損傷後２，４，７，１４日の樹状細胞移植マウスについて、２％パラフォルムアルデヒドで経心臓的灌流固定を行い、凍結切片を作製した（ｎ＝３）。コントロールとして、ＲＰＭＩ１６４０移植群を用いた（ｎ＝３）。次に、一次抗体として抗マウスＭａｃ−１抗体（Ｐｈａｒｍｉｎｇｅｎ社）を利用した免疫組織染色を行った。計測領域に関しては、細胞を移植する際に用いたｇｅｌｆｏａｍ（変性コラーゲン）の最も遠位部、及びそこから１ｍｍ離れた地点それぞれで、背側から腹側に至る部分として、損傷辺縁部、頭側・尾側の３つに分類した。計測するＭａｃ−１陽性細胞の種類に関しては、脊髄外より流入したモノサイト由来のマクロファージと貪食能が特に強い活性型マイクログリアとの両方が含まれるアメーバ状細胞と、貪食能に関しては乏しい活性型マイクログリアと考えられる分枝状細胞の２つに分けて行った。
【００３２】
損傷辺縁部から頭側にかけての代表的切片の染色像を図１１に示す。両群ともに、損傷後２日では細胞浸潤は乏しいが、４日では損傷辺縁部ではアメーバ状細胞の著明な浸潤が認められた。損傷後４日以降では、樹状細胞移植群において、頭側の遠隔部にＭａｃ−１陽性細胞の浸潤が認められたものの、コントロール群ではそのような変化は乏しかった。
【００３３】
次にそれぞれＭａｃ−１陽性細胞を画像解析装置（Ｆｌｏｖｅｌ社）を用いて定量的に解析した。図１２にアメーバ状細胞数の領域別の経時的変化を示す。アメーバ状細胞の浸潤はほとんど損傷辺縁部に限局していた。損傷辺縁部や尾側において両群間で明らかな細胞数の違いを認めなかったが、頭側ではとくに損傷後１４日目に樹状細胞移植群で陽性細胞が多く認められた。一方、図１３は分枝状細胞数の領域別の経時的変化を示しているが、細胞数はすべての領域、計測日で樹状細胞移植群において多い結果となった。樹状細胞移植群で、頭側でアメーバ状の活性型マイクログリアの増加がみられたことは、特にアメーバ状細胞は貪食能が強いため、損傷部から離れた場所で神経軸索伸展を阻害する変性ミエリンや損傷組織に由来する蛋白質の除去を行っていると考えられる。一方、広範囲で分枝状の活性型マイクログリアの増加がみられたことは、活性型マイクログリア自身がＮＴ−３、ＣＮＴＦ、ＩＬ−６、ＴＧＦ−β１、ＥＧＦ、ｂＦＧＦ、ＮＧＦ、ＢＤＮＦ、ＧＤＮＦなどの神経栄養因子を分泌することにより、神経機能の回復を促進させたと考えられる。
【００３４】
実施例１１（樹状細胞移植による内在性神経幹細胞／前駆細胞の解析）
樹状細胞移植による内在性神経幹細胞／前駆細胞の反応性を検討するため、それらを認識するＭｕｓａｓｈｉ−１抗体を用いて、免疫組織染色を行い、陽性細胞数の経時的な変化を調べた。まず、損傷後２、４、７日の樹状細胞移植マウスについて、２％パラフォルムアルデヒドで経心臓的灌流固定を行い、凍結切片を作製した（ｎ＝３）。コントロールとして、ＲＰＭＩ１６４０移植群を用いた（ｎ＝３）。次に、一次抗体として抗マウスＭｕｓａｓｈｉ−１抗体を利用した免疫組織染色を行った。Ｍｕｓａｓｈｉ−１は１９９４年にＯｋａｎｏらにより同定された分子量約３８ｋＤａのＲＮＡ結合タンパクであり（Ｎｅｕｒｏｎ，　１９９４）、マウスのＭｕｓａｓｈｉ−１に対するモノクローナル抗体を用いた解析では神経幹細胞／前駆細胞に強く発現することが報告されている（Ｄｅｖ．　Ｂｉｏｌ．　１９９６，　Ｊ．　Ｎｅｕｒｏｓｃｉ．　１９９７，　Ｄｅｖ．　Ｎｅｕｒｏｓｃｉ．　２０００）。計測領域に関しては、細胞を移植する際に用いたｇｅｌｆｏａｍ（変性コラーゲン）の最も遠位部、及びそこから１ｍｍ離れた地点それぞれで、背側から腹側に至る部分として、損傷辺縁部、遠位部（頭側・尾側）の２つに分類した（図１４）。
損傷辺縁部から頭側にかけての代表的切片の染色像を図１５に示す。両群ともに、損傷後２日では差はみられないが、損傷後４日以降では、樹状細胞移植群において、辺縁部、遠位部ともにＭｕｓａｓｈｉ−１陽性細胞が多く認められたものの、コントロール群ではそのような変化は乏しかった。
【００３５】
次にＭｕｓａｓｈｉ−１陽性細胞を画像解析装置（Ｆｌｏｖｅｌ社）を用いて定量的に解析した。図１６にＭｕｓａｓｈｉ−１陽性細胞数の領域別経時変化を示す。損傷後４日以降で損傷辺縁部や遠位部ともに、コントロールと比較して樹状細胞移植により有意なＭｕｓａｓｈｉ−１陽性細胞数の増加を認めた。特に損傷辺縁部では、損傷後２日から４日の間に樹状細胞移植群でＭｕｓａｓｈｉ−１陽性細胞の著しい増加が認められた。
以上、樹状細胞の移植により、内在性神経幹細胞／前駆細胞が増殖誘導されることが明らかになった。
【００３６】
実施例１２（ＧＭ−ＣＳＦ投与後の損傷神経組織における神経栄養因子の発現誘導）
生後６週齢のＢＡＬＢ／ｃ雌マウスを用いて、脊髄損傷モデルマウスを作成した。損傷直後に、生理食塩水のみ又はＧＭ−ＣＳＦ（２５０ｐｇ／マウス；Ｇｅｎｚｙｍｅ社製）を５μｌ脊髄損傷部位に投与し、２日目に脊髄を摘出した。摘出した脊髄は液体窒素中で凍結後８０℃にて保存し、ＴＲＩｚｏｌ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）を用いて全ＲＮＡを抽出した。各５μｇの全ＲＮＡからＡＭＶ（Ａｖｉａｎ　Ｍｙｅｌｏｂｌａｓｔｏｓｉｓ　Ｖｉｒｕｓ）逆転写酵素とオリゴ（ｄＴ）プライマーを用いて、４２℃で６０分間インキュベートし、総量２００μｌのｃＤＮＡを合成した。β−アクチンのプライマーを用いてＰＣＲを施行し、遺伝子発現を確認した後、各神経栄養因子についてそれぞれの条件下でＰＣＲを行った。ＰＣＲは１μｌのｃＤＮＡをテンプレートとしてＥｘｔａｑ（ＴＡＫＡＲＡ社製）の反応酵素を用いて、サーマルサイクラー（Ｐｅｒｋｉｎ−Ｅｌｍｅｒ社）により遺伝子を増幅した。用いたプライマーとＰＣＲ条件を［表２］に示す。なお混入したゲノミックＤＮＡから増幅された遺伝子産物ではないことを示すために、コントロールとして全ＲＮＡをテンプレートとして、それぞれＰＣＲ反応を行った。
【００３７】
【表２】

【００３８】
損傷脊髄にＧＭ−ＣＳＦを投与することにより、インビボでの神経再生効果やマイクログリアの増殖、食作用の増強を誘導する神経栄養因子ＮＴ−３、損傷脊髄の運動ニューロンの変性、脱落を抑制する神経栄養因子ＢＤＮＦ、コリン作動性ニューロンの神経栄養因子ＮＧＦ、及び脊髄の運動知覚両神経に対し変性・細胞死保護の効果を有する神経栄養因子ＣＮＴＦの発現が誘導されることが明らかになった（図１７）。
【００３９】
実施例１３（ＧＭ−ＣＳＦ投与による内在性マイクログリアの活性化）
ＧＭ−ＣＳＦはインビトロでマイクログリア及びマクロファージの増殖や活性化に関与することが知られているが、中枢神経系組織内のマイクログリア及び損傷部血管から侵入したマクロファージに対する反応性を解析するため、それらを認識するＭａｃ−１抗体を用いて、免疫組織染色を行い、陽性細胞数の経時的な変化を調べた。まず、損傷後２、４、７日のＧＭ−ＣＳＦ投与マウスについて、２％パラフォルムアルデヒドで経心臓的灌流固定を行い、凍結切片を作製した（ｎ＝３）。コントロールとして、生理食塩水投与マウスを用いた（ｎ＝３）。次に、一次抗体として抗マウスＭａｃ−１抗体（Ｐｈａｒｍｉｎｇｅｎ社）を利用した免疫組織染色を行った。計測領域に関しては、細胞を移植する際に用いたｇｅｌｆｏａｍ（変性コラーゲン）の最も遠位部から背側及び腹側１ｍｍ離れた領域を解析した（図１８）。計測するＭａｃ−１陽性細胞の種類に関しては、貧食能が強い活性型マイクログリア及び脊髄外から流入したモノサイト由来のマクロファージと考えられるアメーバ状（ａｍｅｂｏｉｄ）細胞と、貧食能は乏しいが種々の神経栄養因子やサイトカインを分泌する活性型マイクログリアと考えられる分枝状（ｒａｍｉｆｉｅｄ）細胞の２つに分けて、画像解析装置（Ｆｌｏｖｅｌ社）を用いて定量的に解析した。
【００４０】
図１９及び２０にアメーバ状及び分岐状細胞数の経時的変化を示す。ＧＭ−ＣＳＦ投与群では損傷後２日目から多くのアメーバ状細胞を認め、７日目においてコントロールと比較して有意な細胞数の増加を認めた。また、分岐状細胞に関しても、コントロールと比較して損傷後４日目、７日目において有意な細胞数の増加を認めた。ＧＭ−ＣＳＦ投与群でアメーバ状細胞の増加がみられたことは、特にアメーバ状細胞は貧食能が強いため、神経軸索伸展を阻害する変性ミエリンや損傷組織に由来する蛋白質の除去を行っていると考えられる。また分岐状細胞の増加がみられたことは、活性型マイクログリア自身がＮＴ−３、ＢＤＮＦ、ＮＧＦ、ＣＮＴＦ等の神経栄養因子を分泌することにより、神経機能の回復を促進させたと考えられる。
【００４１】
実施例１４（ＧＭ−ＣＳＦ投与による内在性神経幹細胞／前駆細胞の増殖誘導）ＧＭ−ＣＳＦ投与による中枢神経系内の神経幹細胞／前駆細胞に対する反応性を解析するため、それらを認識するＭｕｓａｓｈｉ−１抗体を用いて、免疫組織染色を行い、陽性細胞数の経時的な変化を調べた。まず、損傷後２、４、７日のＧＭ−ＣＳＦ投与マウスについて、２％パラフォルムアルデヒドで経心臓的灌流固定を行い、凍結切片を作製した（ｎ＝３）。コントロールとして、生理食塩水投与マウスを用いた（ｎ＝３）。次に、一次抗体として抗Ｍｕｓａｓｈｉ−１抗体を利用した免疫組織染色を行った。計測領域に関しては、細胞を移植する際に用いたｇｅｌｆｏａｍの最も遠位部から背側及び腹側０．５ｍｍ離れた領域を画像解析装置を用いて定量的に解析した（図２１）。図２２にＭｕｓａｓｈｉ−１陽性細胞数の経時的変化を示す。ＧＭ−ＣＳＦ投与群では損傷後２日目からコントロールと比較して多数のＭｕｓａｓｈｉ−１陽性細胞を認め、７日目において有意な細胞数の増加を認めた。以上、ＧＭ−ＣＳＦ投与により、内在性神経幹細胞／前駆細胞が増殖誘導されることが明らかになった。
【００４２】
以上のことより、損傷部位への樹状細胞移植により、自身の分泌する神経栄養因子、内在性マイクログリアの活性化を介した神経栄養因子の分泌及び神経軸索伸展の阻害因子の除去作用、並びに、内在性神経幹細胞／前駆細胞の増殖誘導による新たな神経細胞の再生及び再髄鞘化などを介して、神経機能が回復されたと考えられる。また、損傷部位へのＧＭ−ＣＳＦ投与により、神経細胞内における神経栄養因子の発現誘導、内在性マイクログリアの活性化を介した神経栄養因子の分泌及び神経軸索伸展の阻害因子の除去作用、並びに、内在性神経幹細胞／前駆細胞の増殖誘導による新たな神経細胞の再生及び再髄鞘化などを介して、神経機能が回復されたと考えられる。
【００４３】
【発明の効果】
本発明の神経損傷又は神経機能不全疾患治療薬は、損傷部位局所に注入するだけでなく、皮下又はリンパ節近傍や静脈内投与を含む様々な投与法が可能であり、優れた神経機能回復作用を有すため、脊髄損傷を含む中枢神経系損傷や脳梗塞等の神経機能不全疾患等の疾患に有用である。また、ＩＬ−１２、ＧＭ−ＣＳＦなどの樹状細胞関連有効物質は、取り扱いが簡便でかつ長期保存が可能で、いつでも大量に調製することができる点で有用であり、遺伝子治療などへの応用が可能である。
【００４４】
【配列表】

【図面の簡単な説明】
【図１】脊髄損傷モデルＢＡＬＢ／ｃマウスの運動機能評価の結果を示す図である。
【図２】脊髄損傷モデルＣ５７ＢＬ／６マウスの運動機能評価の結果を示す図である。
【図３】脊髄損傷に対する樹状細胞を含む抗原提示細胞の効果を示す図である。
【図４】脊髄損傷に対するＣＤ１１ｃ（＋）の樹状細胞の効果を示す図である。
【図５】脊髄損傷に対する本発明のＩＬ−１２の効果を示す図である。
【図６】脊髄損傷に対する本発明のＧＭ−ＣＳＦの効果を示す図である。
【図７】ＲＴ−ＰＣＲによる未成熟樹状細胞サブセットにおける神経栄養因子の発現結果を示す図である。
【図８】ＲＴ−ＰＣＲによる成熟樹状細胞サブセットにおける神経栄養因子の発現結果を示す図である。
【図９】ＥＬＩＳＡによる樹状細胞等のＮＴ−３の分泌結果を示す図である。
【図１０】脊髄損傷に対する神経栄養因子を分泌する樹状細胞サブセットの効果を示す図である。
【図１１】樹状細胞（ＤＣ）、ＲＰＭＩ１６４０（ＲＰＭＩ）移植群それぞれにおける、抗Ｍａｃ−１抗体を用いた免疫染色の結果、特に損傷辺縁部から頭側にかけての経時的な代表的切片を示す写真である。
【図１２】樹状細胞、ＲＰＭＩ１６４０移植群それぞれにおける、Ｍａｃ−１陽性アメーバ状細胞数の領域別の経時的変化を示す図である。
【図１３】樹状細胞、ＲＰＭＩ１６４０移植群それぞれにおける、Ｍａｃ−１陽性分枝状細胞数の領域別の経時的変化を示す図である。
【図１４】Ｍｕｓａｓｈｉ−１陽性細胞数計測のための領域設定を示す写真である。
【図１５】樹状細胞（ＤＣ）、ＲＰＭＩ１６４０（ＲＰＭＩ）移植群それぞれにおける、抗Ｍｕｓａｓｈｉ−１抗体を用いた免疫染色の結果、特に損傷辺縁部から頭側にかけて経時的な代表的切片を示す写真である。
【図１６】樹状細胞、ＲＰＭＩ１６４０移植群それぞれにおける、Ｍｕｓａｓｈｉ−１陽性細胞数の領域別の経時的変化を示す図である。
【図１７】ＲＴ−ＰＣＲによるＧＭ−ＣＳＦ投与後の脊髄損傷部位における神経栄養因子の発現結果を示す図である。
【図１８】Ｍａｃ−１陽性細胞数計測のための領域設定を示す図である。
【図１９】ＧＭ−ＣＳＦ投与群、コントロール（生理食塩水投与）群それぞれにおける内在性マイクログリア細胞（アメーバ状）数の経時的変化示す図である。
【図２０】ＧＭ−ＣＳＦ投与群、コントロール（生理食塩水投与）群それぞれにおける内在性マイクログリア細胞（分岐状）の経時的変化示す図である。
【図２１】Ｍｕｓａｓｈｉ−１陽性細胞数計測のための領域設定を示す図である。
【図２２】ＧＭ−ＣＳＦ投与群、コントロール（生理食塩水投与）群それぞれにおける、Ｍｕｓａｓｈｉ−１陽性細胞数の経時的変化を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a therapeutic agent that promotes nerve regeneration and can be applied to neurological dysfunction diseases such as central nervous system damage including spinal cord injury and cerebral infarction, and particularly applicable to gene therapy.
[0002]
[Prior art]
Most spinal cord injuries are traumatic and are caused by traffic accidents, sports, work accidents, etc., but non-traumatic ones are caused by inflammation, bleeding, tumors, spinal deformities, and the like. The pathological condition is crushing and compression lesions of the spinal cord with hemorrhage in the spinal cord parenchyma and edema as the substrate, and neuropathy corresponding to the damaged site occurs. Major clinical symptoms include failure or complete motor and sensory paralysis below the impairment level, and cervical spinal cord injury includes respiratory paralysis and hyperthermia (or hypothermia) as specific complications. The improvement of the above-mentioned neurological disorders, particularly the improvement of movement disorders, is directly linked to the prevention of bedridden elderly people and the improvement of QOL (Quality of Life), and its importance is increasing along with the extension of the average life span in recent years.
[0003]
As a treatment method for the spinal cord injury, a surgical operation for removing physical compression or injury and a steroid therapy for spinal cord edema in an acute injury stage (see Non-Patent Document 1). ). Among steroids, it has been reported that large doses of methylprednisolone are effective in improving neurological symptoms associated with spinal cord injury (see Non-Patent Document 2), but large doses of steroids have strong systemic side effects. In addition to being difficult to control, there is a problem that spinal cord injury associated with infection causes a decrease in the protective function of infection. Moreover, even the effectiveness of high-dose steroid therapy is currently being discussed. As described above, there is no effective therapeutic agent for spinal cord injury until now, and development of a new therapeutic agent is eagerly desired. Other reported methods of treating spinal cord injury include the use of a therapeutically effective amount of astrocytes pretreated with inflammation-related cytokines in vitro at the site of injury in the central nervous system (CNS). A mammal by administering a transplantation method (see Patent Document 1) or homologous mononuclear phagocytic cells (monocytes, macrophages, etc.) at the site of injury or disease or in the vicinity of the central nervous system (CNS). And a method of promoting nerve axon regeneration in the CNS (see Patent Document 2 and Non-Patent Documents 3 to 6). In addition, although the clear mechanism is unknown, the recovery of motor maintenance after spinal cord injury was promoted by administration of T cells specific for vaccination by spinal cord homogenate and myelin basic protein myelin protein. Has also been reported (see Non-Patent Documents 7 and 8).
[0004]
On the other hand, dendritic cells (DC) are cell populations having a dendritic form derived from hematopoietic stem cells, and are widely distributed in the living body. The immature dendritic cells recognize and take up foreign substances such as viruses and bacteria that have invaded each tissue, and digest and decompose them in the process of migration to the lymphoid organ T-cell region to produce MHC molecules. It plays a role as an antigen-presenting cell that induces an immune response by activating antigen-specific T cells by binding to and presenting on the cell surface (see Non-Patent Documents 9 and 10).
[0005]
Although dendritic cells are widely distributed, the density in each tissue was not high, so it was difficult to prepare a large number of cells, but in vitro by adding differentiation growth factors to immature progenitor cell cultures. In response to the fact that a large number of cells can be easily prepared, the use of dendritic cells as an immunostimulating agent has begun to be studied (see Non-Patent Document 11). In particular, it seeks to specifically enhance the immune response by pulsing antigens into dendritic cells against a weak tumor immune response. In animal experiments, it has been shown that dendritic cells presenting tumor-derived proteins and antigen peptides induce specific CD8 ⁺ cytotoxic T cells. It has been reported that tumors are reduced or eliminated by returning them to living bodies together with dendritic cells. On the other hand, IL-12, which is a cytokine, is secreted mainly from the above-described dendritic cells and B cells, which are antigen-presenting cells, acts on T cells and NK cells, and its high antitumor activity has been reported (Non-patent Document 12). 13). Thus, IL-12 has been attracting attention as a therapeutic agent for cancer, and clinical trials have been conducted as a new immunotherapy for cancer, but it has never been used for the nervous system.
[0006]
On the other hand, motor function evaluation is one of the most important in the study of spinal cord injury using animal models. Such motor function evaluation is desired to be simple and have high reproducibility. However, conventional motor function evaluation methods include the movement of individual joints in the hind limbs, such as the BBB score method (Non-Patent Document 14), which evaluates the walking behavior of animals with a total score (up to 21 points) of various check items, and Many of them focused on their coordinated movements and the overall walking state. Some of them recorded video on the videotape and required detailed measurement after recording. There was a problem that it was likely to occur.
[0007]
[Patent Document 1]
JP 2000-503983 A [Patent Document 2]
Japanese National Patent Publication No. 11-13370 [Non-Patent Document 1]
N. Engl. J. et al. Med. 322, 1405-1411, 1990, J. MoI. Neurosurg 93, 1-7, 2000
[Non-Patent Document 2]
J. et al. Spinal Disorder. 5 (1), 125-131, 1992
[Non-Patent Document 3]
J. et al. Mol. Med. 77, 713-717, 1999
[Non-Patent Document 4]
J. et al. Neurosci. 19 (5), 1708-16, 1999
[Non-Patent Document 5]
Neurosurgery 44 (5), 1041-5, 1999
[Non-Patent Document 6]
Trends. Neurosci 22 (7), 295-9, 1999
[Non-Patent Document 7]
Neuron 24, 639-647, 1999
[Non-Patent Document 8]
Lancet 354, 286-287, 2000
[Non-patent document 9]
Ann. Rev. Immunol. 9, 271-296, 1991
[Non-Patent Document 10]
J. et al. Exp. Med. 185, 2133-2141, 1997
[Non-Patent Document 11]
J. et al. Exp. Med. 183, 7-11, 1996
[Non-Patent Document 12]
J. et al. Exp. Med. 178, 1223-1230, 1993
[Non-Patent Document 13]
J. et al. Exp. Med. 189, 1121-1128, 1999
[Non-Patent Document 14]
J Neurosung 93, 266-75, 2000
[0008]
[Problems to be solved by the invention]
Injuries to the central nervous system, including spinal cord injury, are extremely difficult to treat. As described above, there is no effective treatment to date, and the development of a new treatment is strongly desired. As the population ages, the number of patients suffering from nervous system diseases tends to increase, which is a major social problem. However, the central nervous system is an organ that is extremely difficult to regenerate and is a special organ that is unlikely to cause an immune reaction. In the above-mentioned method of promoting nerve axon regeneration of the central nervous system (CNS) using macrophages by Schwartz et al., It was not clear what functions of macrophages act on axon regeneration. In addition, when cells such as macrophages are used, there is a problem that not only the administration method is limited, but the handling is complicated and it is difficult to obtain a reproducible therapeutic effect only by using living cells. The object of the present invention is not only to inject locally into the damaged site, but also to various administration methods including subcutaneous or near lymph node and intravenous administration, easy handling and long-term storage, It is an object of the present invention to provide a therapeutic agent for neurological dysfunction diseases such as central nervous system injury and cerebral infarction including spinal cord injury having an excellent nerve regeneration promoting action, which can be prepared.
[0009]
[Means for Solving the Problems]
Unlike other tissues, the central nervous system is isolated from the immune system. Recently, however, the present inventors have conducted experiments using a mouse brain tumor model that immature T cells that have not been stimulated cannot invade the central nervous system, but are activated by antigens in the brain. Have reported that they can cross the blood brain barrier and react with brain tumors (Neuro-Oncology 1, S105, 1999). In addition, there is a report that administration of nerve-specific T cells promoted functional recovery of central nerve damage (Lancet 354, 286-287, 2000). Whether nerve-specific T cells function through the blood-brain barrier and function in the central nervous system through the release of some cytokines or do they act directly on nerve cells and axons? Although it is still unknown, the possibility of nerve regeneration by immune system intervention has been shown. On the other hand, in order to induce nerve-specific T cells, it is necessary to take up antigens of the nervous system by antigen-presenting cells and present antigen peptides processed in the cells to T cells.
[0010]
The present inventors have shown that the elimination of damaged tissue during spinal cord injury is an extremely important first step, and a specific subset of dendritic cells that take up antigen and have the highest antigen-presenting ability to T cells are extracted from spinal cord injury model mice. For the first time, it has been demonstrated that restoration of spinal cord function is facilitated by direct transplantation to the site of injury. In order to demonstrate the above-described promotion of recovery of spinal cord function, the method for evaluating motor function in spinal cord injury mice established by the present inventors was used. This motor function evaluation method is an application of a device used for measurement of momentum for the purpose of analyzing the sedative effect of drugs to the evaluation of motor function after spinal cord injury. The present inventors target substances that are secreted from dendritic cells that cause environmental changes in the central nervous system including T cell activation, and substances that induce, proliferate or activate dendritic cells, When administered to the injury site of a spinal cord injury model mouse and screened by the method for evaluating motor function in the above spinal cord injury mouse, IL-12 or IL-12 which is widely used as a therapeutic agent for cancer but not used at all in the nervous system , GM-CSF was found to promote recovery of spinal cord function as well as dendritic cells. In addition, as mentioned above, significant denaturation of motor function was observed by transplanting dendritic cell subsets into the injured spinal cord, so we analyzed substances that stimulate nerve regeneration secreted from dendritic cells. As a result, it was confirmed that dendritic cells expressed and actually secreted neurotrophic factor. The present invention has been completed based on these findings.
[0011]
That is, the present invention relates to a substance secreted from dendritic cells, a substance that induces and proliferates dendritic cells, a substance that activates dendritic cells, a substance that induces expression of neurotrophic factors in neural tissue, and neural tissue One or more substances selected from substances that induce and proliferate microglia, macrophages, or dendritic cells as an active ingredient, a therapeutic agent for nerve injury or dysfunction (claim) 1), substances secreted from dendritic cells, substances that induce and proliferate dendritic cells, substances that activate dendritic cells, substances that induce the expression of neurotrophic factors in neural tissue, and neural tissues The agent for inducing or proliferating microglia or macrophages is a cytokine, which is a therapeutic agent for nerve injury or neurological dysfunction (Claim 2) or secreted from dendritic cells. The therapeutic agent for nerve damage or neurological dysfunction according to claim 2, wherein the cytokine is interleukin (IL) -12, or the cytokine that induces / proliferates dendritic cells, It is granulocyte-macrophage colony-stimulating factor (GM-CSF), The therapeutic agent for nerve damage or neurological dysfunction according to claim 2 (claim 4), or the expression of neurotrophic factor in nerve tissue The therapeutic agent for nerve damage or neurological dysfunction according to claim 2 (Claim 5), or the microbe in neural tissue, wherein the cytokine to be induced is granulocyte-macrophage colony stimulating factor (GM-CSF). Glia, a cytokine that induces and proliferates macrophages is granulocyte-macrophage colony stimulating factor (GM-CSF) The therapeutic agent for nerve damage or neurological dysfunction according to claim 2 (claim 6), a substance secreted from dendritic cells, a substance that induces and proliferates dendritic cells, and a substance that activates dendritic cells 7. The therapeutic agent for nerve injury or neurological dysfunction according to claim 1, wherein one or more selected substances are vectors capable of expressing the substances (claim 7). .
[0012]
Further, in the present invention, the dendritic cell is a dendritic cell subset secreting the neurotrophic factor NT-3, The therapeutic agent for nerve injury or neurological dysfunction according to claim 1 (claim 8) A subset of dendritic cells secreting neurotrophic factor NT-3, in addition to NT-3, an immature dendritic cell subset expressing CNTF, TGF-β1, IL-6, or NT-3, The therapeutic agent for nerve injury or neurological dysfunction according to claim 8, which is a subset of mature dendritic cells expressing CNTF, TGF-β1, IL-6, EGF, or neurotrophic factor (9) The dendritic cell subset secreting NT-3 is an immature dendritic cell subset having a surface marker of CD11c on the cell surface or a mature dendritic cell subset derived from the immature dendritic cell, The therapeutic agent for nerve injury or neurological dysfunction according to claim 8 or 9, wherein the mature dendritic cell subset is immature in the presence of a stimulant for maturation of immature dendritic cells. The therapeutic agent for nerve injury or neurological dysfunction according to claim 9 or 10 (claim 11), or a mature tree, which is a mature dendritic cell subset obtained by culturing a dendritic cell subset in vitro 12. The nerve injury or the nerve dendritic cell according to any one of claims 9 to 11, wherein the dendritic cell subset is a mature dendritic cell subset into which an expression system of a nervous system protein or peptide or a gene encoding the same is introduced. A therapeutic agent for neurological dysfunction (Claim 12) or a therapeutic agent for nerve injury or neurological dysfunction according to any one of Claims 1 to 12, wherein Lymph node near, or to a method of treating nerve damage or nerve dysfunctional disorder which comprises administering intravenous (Claim 13).
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Examples of the therapeutic agent for nerve injury or neurological dysfunction according to the present invention include substances secreted from dendritic cells, substances that induce and proliferate dendritic cells, substances that activate dendritic cells, Substance that induces expression of neurotrophic factor, substance that induces and proliferates microglia and nerve macrophages in nerve tissue, and has a preventive effect, symptom improvement effect or therapeutic effect on nerve damage or neurological dysfunction disease ( Hereinafter, these substances are collectively referred to as “dendritic cell-related active substances”), and those containing a mixture of these substances as active ingredients. The substances secreted from the dendritic cells include IL-12, IL-1α, IL-1β, IFN-γ and other cytokines can be preferably exemplified, and as a substance that induces and proliferates dendritic cells, GM-CSF, IL-4 and other cytokines As a substance that activates dendritic cells, IL-1β, CD40L and the like can be preferably exemplified, and a substance that induces expression of neurotrophic factor in nerve tissue after injury Can be preferably exemplified by cytokines such as GM-CSF, and as a substance that induces and proliferates microglia and nerve macrophages in damaged nerve tissues, cytokines such as GM-CSF and M-CSF are preferably exemplified. can do. Examples of the neurotrophic factor include in vivo nerve regeneration effect, microglial proliferation, NT-3 which induces enhancement of phagocytosis, BDNF which suppresses degeneration and loss of motor neurons in injured spinal cord, nerves of cholinergic neurons Examples thereof include trophic factor NGF and CNTF, which has an effect of protecting against degeneration and cell death against both motor sensory nerves of the spinal cord.
[0014]
Substances secreted from dendritic cells, substances that induce and proliferate dendritic cells, substances that activate dendritic cells, substances that induce the expression of neurotrophic factors in neural tissues, As the substance that induces and proliferates microglia and macrophages, each known substance having an action of inducing and proliferating dendritic cells can be used. For example, substances secreted from dendritic cells include dendritic cells. Can be obtained by in vitro culturing, and a substance having an action of inducing and proliferating dendritic cells can be obtained by culturing dendritic cells in vitro in the presence of a candidate substance, The substance that activates dendritic cells can be obtained by measurement and evaluation. The dendritic cells are cultured in vitro in the presence of a candidate substance, and the degree of neurotrophic factor production ability of the dendritic cells is measured / evaluate The substance that induces the expression of neurotrophic factor in nerve tissue can be obtained by measuring and evaluating the degree of expression and induction of neurotrophic factor in damaged nerve tissue to which the candidate substance is administered. The substance that induces and proliferates microglia and macrophages in the nerve tissue is derived from the activated microglia with strong phagocytosis and monosite that flows from outside the spinal cord in the damaged nerve tissue to which the candidate substance is administered The degree of induction / proliferation of ameboid cells considered to be macrophages of the human body, and branched cells considered to be active microglia that secrete various neurotrophic factors and cytokines, although poor phagocytosis It can be obtained by measuring and evaluating.
[0015]
When the above dendritic cell-related active substance is used as a therapeutic agent for nerve injury or neurological dysfunction, a pharmaceutically acceptable carrier, binder, stabilizer, excipient, diluent, pH buffer, Various preparation ingredients such as a disintegrant, a solubilizer, a solubilizer, and an isotonic agent can be added. Such therapeutic agents can be administered orally or parenterally. That is, it can be administered orally in commonly used dosage forms, such as powders, granules, capsules, syrups, suspensions, etc., or, for example, in dosage forms such as solutions, emulsions, suspensions, etc. In addition to the topical administration parenterally in the form of injection, it can also be administered intranasally in the form of a spray.
[0016]
Further, as the dendritic cell-related active substance, a vector capable of expressing the substance can be used. When such a vector is locally administered as gene therapy, a treatment using the dendritic cell-related active substance as an active ingredient Compared to when the drug is administered locally, stable expression of the substance makes it possible to stably supply the dendritic cell-related active substance locally. Many dendritic cell-related active substances have very short half-lives and are unstable, whereas genes that can express dendritic cell-related active substances are used to introduce genes into cells at the site of nerve injury Thus, stable expression for a predetermined period can be obtained. Suitable examples of such vectors include viral vectors such as herpes virus (HSV) vectors, adenovirus vectors, and human immunodeficiency virus (HIV) vectors. Among these viral vectors, HSV vectors are preferred. HSV vectors have high neurophilicity, are safe because HSV is not integrated into the chromosomal DNA of cells, and can regulate the expression period of the transgene. Moreover, the viral vector which expresses a dendritic cell related active substance can be prepared by a conventional method.
[0017]
Examples of the therapeutic agent for nerve damage or neurological dysfunction according to the present invention include dendritic cells, particularly preferably those having dendritic cell subsets secreting neurotrophic factor NT-3 as active ingredients. Dendritic cell subsets that secrete the neurotrophic factor NT-3 include in vivo nerve regeneration, microglial proliferation and phagocytosis, in addition to NT-3 that induces degeneration of spinal motor sensory neurons. CNTF, which shows the effect of protecting cell death, TGF-β1 which has an action of suppressing the release of cytotoxic substances derived from microglia and macrophages, and IL- which induces the protective effect on various neurons (choline / catecholamine / dopaminergic) In addition to immature dendritic cell subset expressing NT6, NT-3, CNTF, TGF-β1, IL-6, neuroprotective effect Recognized mature dendritic cell subsets that express EGF are preferred, and examples include immature dendritic cell subsets that have a CD11c surface marker on the cell surface and mature dendritic cell subsets derived from the immature dendritic cells. Can do.
[0018]
Then, the immature dendritic cell subset is cultured in vitro in the presence of a stimulant for maturation of immature dendritic cells such as LPS, IL-1, TNF-α, and CD40L as the mature dendritic cell subset. The mature dendritic cell subset obtained by doing can also be used. In this case, the expression of neurotrophic factors such as NT-3 may change, and a higher regeneration effect may be induced. In addition, a protein or peptide of the nervous system such as a myelin protein such as MBP (myelin basic protein) or MAG (myelin-associated glycoprotein), a factor that suppresses the extension of nerve axons such as Nogo, or a gene encoding them is It is also possible to use mature dendritic cell subsets into which (incorporated) an expression system such as a virus vector that has been introduced.
[0019]
Dendritic cell subsets secreting neurotrophic factor NT-3 are sorted by FACS using a monoclonal antibody against dendritic cell surface antigens after, for example, pretreatment such as density centrifugation for peripheral blood and the like By separating a dendritic cell subset by a method or a separation method using a magnetic bead-binding monoclonal antibody against a dendritic cell surface antigen, and selecting a dendritic cell subset secreting NT-3 from them it can. The dendritic cell subset secreting such neurotrophic factor NT-3 can be transplanted to a nerve injury site such as spinal cord. In particular, mature dendritic cell subsets into which (incorporated) the expression system for the above-mentioned nervous system proteins or peptides or genes encoding them can be administered subcutaneously or in the vicinity of lymph nodes. As described above, as a method for treating nerve damage or neurological dysfunction disease of the present invention, a nerve comprising the same dendritic cell subset secreting the above-mentioned dendritic cell-related active substance and neurotrophic factor NT-3 as an active ingredient. Examples thereof include a method of administering (transplanting) a therapeutic agent for an injured or neurological dysfunction disease at the site of nerve injury, subcutaneously or near a lymph node, or intravenously.
[0020]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
Example 1 (Preparation of spinal cord injury model BALB / c mouse)
A 6-week-old BALB / c female mouse (n = 9) was used, and the 8th thoracic vertebral arch was excised under ether anesthesia, and the spinal cord was cut into the left half with a sharp blade (injury group; ◇ ) Was produced. After spinal cord injury, the mouse exhibited left lower limb paralysis. As a control (control group; □), a 6-week-old BALB / c female mouse (n = 9) that had undergone only laminectomy was used. After the operation, the spontaneous momentum of each mouse on days 2, 4 and 7 for the acute phase, day 7 for the subacute phase, and days 14, 21, 28 and 56 for the chronic phase was measured using the behavior analysis device SCANETMV-10 (Toyo Sangyo). An apparatus having 144 pairs of near-infrared sensors that run vertically and horizontally in a square of 426 mm square was measured using a two-tiered apparatus) to evaluate motor function. In addition, as the measurement of the spontaneous momentum, the horizontal movement (Moving 1, 2; M1, M2 is abbreviated as large and small), M1 is considered to be operated when there is a movement of 12 mm or more, and M2 is 60 mm or more. Measured) and detected in the form of vertical movement (Rearing; abbreviated as RG, measuring the number of rising motions of 6.75 cm or more) and set to measure for 10 minutes per animal. The results when BALB / c female mice are used are shown in FIG. The p value in the figure was calculated using Student's t test (*: p <0.05, **: p <0.01). As a result of comparison of each motor function evaluation between the control group and the injury group, M1 (upper part of FIG. 1) and M2 (middle part of FIG. 1) showing the motor evaluation in the horizontal direction showed a significant difference in the acute phase and the subacute phase. Although it was observed, there was no significant difference in the chronic phase. On the other hand, in the RG showing the motion evaluation in the vertical direction, a clear significant difference was recognized between the two groups until the chronic stage (lower part of FIG. 1).
[0021]
Example 2 (Production of spinal cord injury model C57BL / 6 mice)
Moreover, it replaced with the said 6-week-old BALB / c female mouse (n = 9), and the behavioral-analysis apparatus similarly to Example 1 except using a C57BL / 6 female mouse (n = 16) 6-week-old. Motor function evaluation was performed using SCANETTMV-10. The results are shown in FIG. In addition, p value in the figure was calculated using Student's t test (**: p <0.01). As a result of comparison of each motor function evaluation between the control group (□) and the injury group (◇), M1 (upper part of FIG. 2), M2 (upper row in FIG. 2) showing the motor evaluation in the horizontal direction throughout the acute phase, subacute phase, and chronic phase. In the middle part of FIG. 2, no obvious significant difference was observed. On the other hand, in the RG showing the motion evaluation in the vertical direction, a clear significant difference was recognized between the two groups until the chronic stage (lower part of FIG. 2). From the above experimental results of two different strains of mice, the horizontal momentum (M1, M2) was compensated by the lower limbs and both upper limbs of the healthy side, and the paralysis of the left lower limb could not be accurately evaluated, whereas the vertical direction It has been shown that exercise (RG) can accurately assess motor function after spinal cord injury.
[0022]
Example 3 (Effect of dendritic cells on spinal cord injury)
A spinal cord injury model mouse (BALB / c female mouse) was prepared by the same operation as in Example 1, and immediately RPMI1640 medium alone [control (◇), FIG. 3; n = 14, FIG. 4; n = 6], or By sorting antigen-presenting cells including dendritic cells isolated from spleen [5 × 10 ⁵ cells / mouse, n = 13 (FIG. 3; ◯)] or a subset of CD11c (+) by immunomagnetic bead method The obtained dendritic cells [1 × 10 ⁵ cells / mouse, n = 6 (FIG. 4; ◯)] were transplanted to the spinal cord injury site. Moreover, the mouse | mouth which performed only the laminectomy was used as a control which does not add spinal cord injury [FIG. 3; □ (n = 6)]. In the same manner as in Example 1, the spontaneous exercise amount in the vertical direction of each mouse on days 2, 4, 7, 14, 21, 28, and 56 was measured using the behavior analysis apparatus SCANETTMV-10, and motor function evaluation was performed. The results are shown in FIGS. The p value in the figure was calculated using Student's t test (*: p <0.05, **: p <0.01). From these results, by administering the CD11c (+) dendritic cell subset to the injury site, a significant difference was observed in the vertical momentum as compared with the control. From the above, it became clear that the recovery of spinal cord function is promoted by administering dendritic cells to the site of nerve injury.
[0023]
Example 4 (Effect of IL-12 on spinal cord injury)
Using 6-week-old BALB / c female mice aged in the same manner as in Example 1, spinal cord injury model mice were prepared. In addition, BALB / c female mice of 6 weeks old (□; n = 6), in which only laminectomy was performed, were used as a control without adding spinal cord injury. Immediately after spinal cord injury, 5 μl of saline alone (◇; n = 14) or IL-12 (100 ng / mouse; Pharmingen, ○; n = 14) was administered to the site of spinal cord injury. In the same manner as above, the spontaneous movement amount in the vertical direction of each mouse on days 2, 4, 7, 14, 21, and 28 was measured using the behavior analysis apparatus SCANETTMV-10, and the motor function was evaluated. The result is shown in FIG. The p value in the figure was calculated using Student's t test (*: p <0.05, **: p <0.01). From these results, by administering IL-12 to the site of injury, a clear significant difference was observed in the amount of exercise in the vertical direction as compared with the administration of physiological saline. From the above, it became clear that administration of IL-12 at the site of nerve injury promotes recovery of spinal cord function as in the case of using the dendritic cells.
[0024]
Example 5 (Effect of GM-CSF on spinal cord injury)
Using 6-week-old BALB / c female mice aged in the same manner as in Example 1, spinal cord injury model mice were prepared. In addition, BALB / c female mice of 6 weeks old (□; n = 6), in which only laminectomy was performed, were used as a control without adding spinal cord injury. Immediately after spinal cord injury, 5 μl of saline alone (◇; n = 7) or GM-CSF (10 ng / mouse; Genzyme, ○; n = 6) was administered to the spinal cord injury site, and the same as in Example 1 In addition, the spontaneous exercise amount in the vertical direction of each mouse on days 2, 4, 7, 14, 21, and 28 was measured using the behavior analysis apparatus SCANETTMV-10 to evaluate the motor function. The result is shown in FIG. In addition, p value in the figure was calculated using Student's t test (**: p <0.01). From these results, a clear significant difference was observed in the amount of exercise in the vertical direction when GM-CSF was administered to the site of injury compared to the administration of physiological saline. From the above, it was revealed that the administration of GM-CSF to the nerve injury site promotes the recovery of spinal cord function as in the case of using the dendritic cells.
[0025]
Example 6 (Preparation of immature dendritic cell subset and mature dendritic cell subset)
Immature dendritic cells were obtained by isolating CD11c positive subsets from the spleen of 6-week-old BALB / c female mature mice by immunomagnetic bead method. Specifically, first, the spleen was homogenized with 100 U / ml collagenase (Worthington Biochemical Corporation), and then the hard-to-separate coating was further treated with 400 U / ml collagenase at 37 ° C. under 5% CO ₂ for 20 minutes. Incubate to separate the cells. The obtained cells were suspended in a 35% BSA solution, and further RPMI 1640 + 10% fetal serum was overlaid in a centrifuge tube, and then centrifuged at 4 ° C., 3000 rpm, 30 minutes, and 35% BSA solution and RPMI 1640 + 10% fetal serum solution The cells in the boundary layer were collected. Next, the obtained cells were reacted with a magnetic bead-bound monoclonal antibody against CD11c antigen (2 × 10 ⁸ beads, Miltenyi Biotec) for 15 minutes at 4 ° C., and the bead-bound cells were separated magnetically to immature. A fraction enriched in dendritic cell subsets was obtained. Moreover, the mature dendritic cell subset was obtained by culturing the obtained immature dendritic cell subset in a culture solution of RPMI 1640 + 10% fetal serum at 37 ° C. under 5% CO _{2 for} 24 hours.
[0026]
Example 7 (Neurotrophic factor gene expression in dendritic cells)
Total RNA was extracted from each cell of the immature dendritic cell subset and mature dendritic cell subset using TRIzol (Life Technologies), and AMV (Avian Myeloblastosis Virus) reverse transcriptase and oligo (oligo) from each 5 μg of total RNA. dT) The primer was used and incubated at 42 ° C. for 60 minutes to synthesize a total amount of 200 μl of cDNA. PCR was performed using a β-actin primer, and after confirming gene expression, PCR was performed under the respective conditions for each neurotrophic factor. In PCR, a gene was amplified by a thermal cycler (Perkin-Elmer) using Extaq (TAKARA) as a template with 1 μl of cDNA as a template. The primers and PCR conditions used are shown in [Table 1]. In order to show that the product was not a gene product amplified from the mixed genomic DNA, PCR was performed using total RNA as a template as a control. The results for the immature dendritic cell subset are shown in FIG. 7, and the results for the mature dendritic cell subset are shown in FIG.
[0027]
[Table 1]

[0028]
In immature dendritic cells, NT-3 induces nerve regeneration effects, microglial proliferation and enhancement of phagocytosis in vivo, CNTF and microglia have effects of degeneration and cell death protection for both spinal motor sensory nerves. Furthermore, expression of IL-6 that induces protective effects on TGF-β1 and various neurons (choline / catecholamine / dopaminergic activity) having an inhibitory action on the release of cytotoxic substances derived from macrophages was confirmed (FIG. 7). Moreover, in mature dendritic cells, in addition to NT-3, CNTF, TGF-β1, and IL-6, expression of EGF, which is recognized to have a neuroprotective effect, was confirmed (FIG. 8). For each gene, cDNA was extracted from the gel and the nucleotide sequence was analyzed, and it was confirmed that the expression products were NT-3, CNTF, TGF-β1, IL-6 and EGF, respectively.
[0029]
Example 8 (secretion of neurotrophic factor NT-3)
Further, NT-3 immunoassay system (Promega) was used to determine whether NT-3, which is one of the most important neurotrophic factors for nerve regeneration, is actually secreted from dendritic cells. Analysis was performed by ELISA. In the same manner as in Example 1, CD11c-positive immature dendritic cells were separated from the spleen of a 6-week-old BALB / c female mature mouse by immunomagnetic bead method. After 1 × 10 ⁵ CD11c-positive immature dendritic cells were incubated in RPMI1640 + 10% fetal serum culture solution at 37 ° C. under 5% CO _{2 for} 24 hours, the culture supernatant was recovered. As a control, only RPMI 1640, and 1 × 10 ⁵ CD4 positive T cells and CD8 positive T cells were used. As a result of quantitative analysis of NT-3 in the supernatant by sandwich ELISA using two kinds of anti-NT-3 antibodies, 1 × 10 ⁵ dendritic cells were found to have about 1.75 ng of NT- in 24 hours. 3 was revealed to be secreted. Secretion was not observed in RPMI 1640 alone, CD4-positive T cells, and CD8-positive T cells (FIG. 9).
[0030]
Example 9 (Reconfirmation of the effect of dendritic cells on spinal cord injury)
A 6-week-old BALB / c female mouse was subjected to excision of the 8th thoracic vertebral arch under ether anesthesia, and a spinal cord injury model mouse was prepared in which the left spinal cord was half-cut under a microscope. Immediately after transplanting 1 × 10 ⁶ dendritic cells into the spinal cord injury site (DC, n = 17), using the lower limb motor function evaluation method developed by the present inventors (behavioral analysis device SCANET MV-10, RG score) which automatically analyzes the number of rises, and BBB scale (evaluated between 0-21 points), which is one of the already established methods of evaluating the lower limb motor function. No, 21 points are normal), and the evaluation was performed over time. As controls, RPMI 1640 (RPMI, n = 18) and CD8 positive T cells (T, n = 10) were similarly transplanted into the damaged spinal cord region. The results are shown in FIG. As can be seen from FIG. 10, both of the two evaluation methods (RG score and BBB scale) showed a statistically significantly higher score in the DC transplanted group than in the case of control T cells or RPMI. Therefore, it was reconfirmed that the restoration of spinal cord function was promoted by transplanting a dendritic cell subset secreting neurotrophic factor NT-3 to the site of spinal cord injury.
[0031]
Example 10 (Activation of endogenous microglia by dendritic cell transplantation)
In order to investigate whether dendritic cell transplantation shows changes in the reactivity of endogenous microglia or macrophages invading from damaged blood vessels, immunohistochemical staining was performed using a Mac-1 antibody that recognizes them. The change in the number of positive cells over time was examined. First, transcardiac perfusion fixation was performed with 2% paraformaldehyde on dendritic cell transplanted mice 2, 4, 7, and 14 days after injury, and frozen sections were prepared (n = 3). As a control, the RPMI1640 transplant group was used (n = 3). Next, immunohistochemical staining was performed using an anti-mouse Mac-1 antibody (Pharmingen) as the primary antibody. Regarding the measurement area, the most distal part of gelfoam (denatured collagen) used when transplanting the cells, and the part from the dorsal side to the ventral side at a point 1 mm away from it, It was classified into three, side and caudal. Regarding the types of Mac-1 positive cells to be measured, amoeboid cells containing both monosite-derived macrophages flowing from outside the spinal cord and active microglia with particularly strong phagocytic activity, and poor active type with respect to phagocytic ability It was divided into two branched cells thought to be microglia.
[0032]
FIG. 11 shows a stained image of a representative section from the damaged marginal part to the head side. In both groups, cell infiltration was poor on the 2nd day after injury, but on the 4th day, there was marked infiltration of amoeba cells on the margin of the injury. After 4 days after injury, in the dendritic cell transplantation group, infiltration of Mac-1 positive cells was observed in the remote part of the head, but such change was scarce in the control group.
[0033]
Next, each Mac-1-positive cell was quantitatively analyzed using an image analyzer (Flovel). FIG. 12 shows time-dependent changes in the number of amoeba cells by region. Infiltration of amoeboid cells was almost confined to the damaged margin. Although there was no obvious difference in the number of cells between the two groups at the injured margin and the caudal side, many positive cells were observed in the dendritic cell transplantation group particularly on the 14th day after injury on the cranial side. On the other hand, FIG. 13 shows the time-dependent changes in the number of branched cells by region, but the number of cells was large in the dendritic cell transplantation group in all regions and measurement days. In the dendritic cell transplantation group, the increase in cranial amoebic active microglia was observed, especially because amoeboid cells have strong phagocytic ability and inhibited nerve axon extension at a location away from the damaged area It is considered that proteins derived from denatured myelin and damaged tissues are removed. On the other hand, an increase in a wide range of branched active microglia was observed because the active microglia itself was NT-3, CNTF, IL-6, TGF-β1, EGF, bFGF, NGF, BDNF, GDNF. It is thought that the recovery of nerve function was promoted by secreting such neurotrophic factors.
[0034]
Example 11 (Analysis of endogenous neural stem / progenitor cells by dendritic cell transplantation)
In order to examine the reactivity of endogenous neural stem cells / progenitor cells by dendritic cell transplantation, immunohistochemical staining was performed using Musashi-1 antibody that recognizes them, and changes in the number of positive cells over time were examined. First, transcardiac perfusion fixation was performed with 2% paraformaldehyde on dendritic cell transplanted mice 2, 4, and 7 days after injury, and frozen sections were prepared (n = 3). As a control, the RPMI1640 transplant group was used (n = 3). Next, immunohistochemical staining using anti-mouse Musashi-1 antibody as a primary antibody was performed. Musashi-1 is an RNA binding protein with a molecular weight of about 38 kDa identified by Okano et al. In 1994 (Neuron, 1994), and is strongly expressed in neural stem / progenitor cells when analyzed with a mouse monoclonal antibody against Musashi-1 (Dev. Biol. 1996, J. Neurosci. 1997, Dev. Neurosci. 2000). As for the measurement area, the most distal part of gelfoam (denatured collagen) used for transplanting the cells and the part 1 mm away from the distal part from the dorsal side to the ventral side, It was classified into two parts (the head side and the caudal side) (FIG. 14).
FIG. 15 shows a stained image of a representative section from the damaged marginal part to the head side. In both groups, no difference was observed on the 2nd day after the injury, but after 4 days after the injury, many dendritic cell transplantation groups showed many Musashi-1 positive cells in both the marginal part and the distal part. Such changes were scarce in the control group.
[0035]
Next, Musashi-1 positive cells were quantitatively analyzed using an image analyzer (Flovel). FIG. 16 shows the time-dependent changes in the number of Musashi-1 positive cells by region. From 4 days after the injury, a significant increase in the number of Musashi-1-positive cells was observed by dendritic cell transplantation compared to the control in both the marginal area and the distal area. In particular, at the margin of injury, a marked increase in Musashi-1-positive cells was observed in the dendritic cell transplant group between 2 and 4 days after injury.
As described above, it has been clarified that dendritic cell transplantation induces proliferation of endogenous neural stem / progenitor cells.
[0036]
Example 12 (Expression induction of neurotrophic factor in damaged nerve tissue after GM-CSF administration)
A spinal cord injury model mouse was prepared using a 6-week-old BALB / c female mouse. Immediately after the injury, 5 μl of saline alone or GM-CSF (250 pg / mouse; manufactured by Genzyme) was administered to the site of spinal cord injury, and the spinal cord was excised on the second day. The excised spinal cord was frozen in liquid nitrogen and stored at 80 ° C., and total RNA was extracted using TRIzol (manufactured by Life Technologies). Each 5 μg of total RNA was incubated with AMV (Avian Myeloblastosis Virus) reverse transcriptase and oligo (dT) primer for 60 minutes at 42 ° C. to synthesize a total amount of 200 μl of cDNA. PCR was performed using a β-actin primer, and after confirming gene expression, PCR was performed under the respective conditions for each neurotrophic factor. In PCR, a gene was amplified by a thermal cycler (Perkin-Elmer) using a reaction enzyme of Extaq (manufactured by TAKARA) using 1 μl of cDNA as a template. The primers and PCR conditions used are shown in [Table 2]. In order to show that the product was not a gene product amplified from the mixed genomic DNA, PCR was performed using total RNA as a template as a control.
[0037]
[Table 2]

[0038]
Administration of GM-CSF to the injured spinal cord suppresses neurotrophic factor NT-3, which induces nerve regeneration effects, microglia proliferation and phagocytosis in vivo, and degeneration and loss of motor neurons in the injured spinal cord It was revealed that the expression of neurotrophic factor BDNF, cholinergic neuron neurotrophic factor NGF, and neurotrophic factor CNTF, which has the effect of protecting against degeneration and cell death, in both spinal motor sensory neurons ( FIG. 17).
[0039]
Example 13 (Activation of endogenous microglia by GM-CSF administration)
GM-CSF is known to be involved in the proliferation and activation of microglia and macrophages in vitro, but in order to analyze the reactivity to microglia and macrophages invading from damaged blood vessels in the central nervous system tissue, Using a Mac-1 antibody that recognizes them, immunohistochemical staining was performed, and changes in the number of positive cells over time were examined. First, GM-CSF-administered mice 2, 4, and 7 after injury were transcardially perfused and fixed with 2% paraformaldehyde to prepare frozen sections (n = 3). As a control, physiological saline-administered mice were used (n = 3). Next, immunohistochemical staining was performed using an anti-mouse Mac-1 antibody (Pharmingen) as the primary antibody. With respect to the measurement region, a region 1 mm away from the most distal portion of gelfoam (denatured collagen) used when cells were transplanted was analyzed (FIG. 18). Regarding the types of Mac-1 positive cells to be measured, active microglia with strong phagocytosis and ameboid cells considered to be monosite-derived macrophages that flowed in from outside the spinal cord, and poor phagocytosis are various. It was divided into two types of branched microglia that are considered to be active microglia secreting various neurotrophic factors and cytokines, and quantitatively analyzed using an image analyzer (Flovel).
[0040]
19 and 20 show changes over time in the number of amoebic and branched cells. In the GM-CSF administration group, many amoeba-like cells were observed from the second day after injury, and a significant increase in the number of cells was observed on the seventh day compared with the control. Further, regarding the branched cells, a significant increase in the number of cells was observed on the 4th and 7th days after the injury as compared with the control. In the GM-CSF administration group, the increase in amoeboid cells was observed, especially because amoeboid cells have strong phagocytic activity, so that demyelin that inhibits nerve axon extension and proteins derived from damaged tissues are removed. It is thought that. The increase in the number of branched cells is thought to be due to the fact that activated microglia themselves secrete neurotrophic factors such as NT-3, BDNF, NGF, and CNTF, thereby promoting the recovery of nerve function.
[0041]
Example 14 (Induction of proliferation of endogenous neural stem cells / progenitor cells by administration of GM-CSF) In order to analyze the reactivity to neural stem cells / progenitor cells in the central nervous system by administration of GM-CSF, Musashi-1 which recognizes them Using an antibody, immunohistochemical staining was performed, and changes in the number of positive cells over time were examined. First, GM-CSF-administered mice 2, 4, and 7 after injury were transcardially perfused and fixed with 2% paraformaldehyde to prepare frozen sections (n = 3). As a control, physiological saline-administered mice were used (n = 3). Next, immunohistochemical staining using an anti-Musashi-1 antibody as a primary antibody was performed. Regarding the measurement region, a region separated 0.5 mm from the most distal part of the gelfoam used for transplanting cells from the dorsal side and the ventral side was quantitatively analyzed using an image analyzer (FIG. 21). FIG. 22 shows changes over time in the number of Musashi-1 positive cells. In the GM-CSF administration group, a large number of Musashi-1 positive cells were observed from the second day after injury compared to the control, and a significant increase in the number of cells was observed on the seventh day. As described above, it was revealed that administration of GM-CSF induces proliferation of endogenous neural stem / progenitor cells.
[0042]
Based on the above, transplantation of dendritic cells to the injured site removes the neurotrophic factor secreted by itself, the secretion of neurotrophic factor through the activation of endogenous microglia, and the inhibitory factor of nerve axon extension, In addition, it is considered that the nerve function was restored through regeneration and remyelination of new nerve cells by inducing proliferation of endogenous neural stem / progenitor cells. In addition, administration of GM-CSF to the site of injury induces the expression of neurotrophic factor in nerve cells, secretion of neurotrophic factor through activation of endogenous microglia, and removal of an inhibitor of nerve axon extension, In addition, it is considered that the nerve function was restored through regeneration and remyelination of new nerve cells by inducing proliferation of endogenous neural stem / progenitor cells.
[0043]
【The invention's effect】
The therapeutic agent for nerve injury or neurological dysfunction according to the present invention can be administered not only locally to the site of injury but also various administration methods including subcutaneous or near lymph node and intravenous administration, and has an excellent nerve function recovery action. Therefore, it is useful for diseases such as central nervous system damage including spinal cord injury and neurological dysfunction diseases such as cerebral infarction. In addition, dendritic cell-related active substances such as IL-12 and GM-CSF are useful in that they are easy to handle and can be stored for a long period of time, and can be prepared in large quantities at any time. Is possible.
[0044]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing the results of motor function evaluation of a spinal cord injury model BALB / c mouse.
FIG. 2 shows the results of motor function evaluation of spinal cord injury model C57BL / 6 mice.
FIG. 3 shows the effect of antigen-presenting cells including dendritic cells on spinal cord injury.
FIG. 4 shows the effect of dendritic cells of CD11c (+) on spinal cord injury.
FIG. 5 is a graph showing the effect of IL-12 of the present invention on spinal cord injury.
FIG. 6 is a diagram showing the effect of GM-CSF of the present invention on spinal cord injury.
FIG. 7 shows the results of expression of neurotrophic factor in immature dendritic cell subsets by RT-PCR.
FIG. 8 shows the results of expression of neurotrophic factor in mature dendritic cell subsets by RT-PCR.
FIG. 9 is a view showing the secretion results of NT-3 such as dendritic cells by ELISA.
FIG. 10 shows the effect of dendritic cell subsets secreting neurotrophic factors on spinal cord injury.
FIG. 11 shows the results of immunostaining using an anti-Mac-1 antibody in the dendritic cell (DC) and RPMI 1640 (RPMI) transplantation groups, and particularly representative sections over time from the damaged margin to the head side. It is a photograph shown.
FIG. 12 is a graph showing time-dependent changes in the number of Mac-1 positive amoeboid cells by region in each dendritic cell and RPMI1640 transplantation group.
FIG. 13 is a graph showing time-dependent changes in the number of Mac-1-positive branched cells in each dendritic cell and RPMI1640 transplantation group.
FIG. 14 is a photograph showing region setting for measuring the number of Musashi-1 positive cells.
FIG. 15 shows the results of immunostaining using an anti-Musashi-1 antibody in the dendritic cell (DC) and RPMI 1640 (RPMI) transplantation groups, and particularly representative sections over time from the damaged margin to the head. It is a photograph.
FIG. 16 is a graph showing time-dependent changes in the number of Musashi-1 positive cells in each dendritic cell and RPMI1640 transplantation group.
FIG. 17 is a view showing the expression result of neurotrophic factor in the spinal cord injury site after GM-CSF administration by RT-PCR.
FIG. 18 is a diagram showing region settings for measuring the number of Mac-1 positive cells.
FIG. 19 shows changes over time in the number of endogenous microglia cells (amoeba-like) in each of the GM-CSF administration group and the control (saline administration) group.
FIG. 20 is a graph showing changes over time of endogenous microglia cells (branched) in each of a GM-CSF administration group and a control (saline administration) group.
FIG. 21 is a diagram showing region settings for measuring the number of Musashi-1 positive cells.
FIG. 22 is a graph showing changes over time in the number of Musashi-1 positive cells in each of the GM-CSF administration group and the control (saline administration) group.

Claims (13)

Substances secreted from dendritic cells, substances that induce and proliferate dendritic cells, substances that activate dendritic cells, substances that induce the expression of neurotrophic factors in neural tissue, and microglia in neural tissue, A therapeutic agent for nerve injury or neurological dysfunction characterized by comprising one or more substances selected from substances that induce and proliferate macrophages, or dendritic cells as active ingredients. Substances secreted from dendritic cells, substances that induce and proliferate dendritic cells, substances that activate dendritic cells, substances that induce the expression of neurotrophic factors in neural tissue, and microglia in neural tissue, The therapeutic agent for nerve damage or neurological dysfunction according to claim 1, wherein the substance that induces / proliferates macrophages is a cytokine. The therapeutic agent for nerve injury or neurological dysfunction according to claim 2, wherein the cytokine secreted from dendritic cells is interleukin (IL) -12. The therapeutic agent for nerve damage or neurological dysfunction according to claim 2, wherein the cytokine that induces and proliferates dendritic cells is granulocyte-macrophage colony stimulating factor (GM-CSF). The therapeutic agent for nerve damage or neurological dysfunction according to claim 2, wherein the cytokine that induces the expression of neurotrophic factor in nerve tissue is granulocyte-macrophage colony stimulating factor (GM-CSF). The therapeutic agent for nerve damage or neurological dysfunction according to claim 2, wherein the cytokine that induces and proliferates microglia and macrophages in nerve tissue is granulocyte-macrophage colony stimulating factor (GM-CSF). A vector capable of expressing one or more substances selected from substances secreted from dendritic cells, substances that induce and proliferate dendritic cells, and substances that activate dendritic cells. The therapeutic agent for nerve damage or neurological dysfunction according to claim 1, wherein The therapeutic agent for nerve injury or neurological dysfunction according to claim 1, wherein the dendritic cell is a dendritic cell subset secreting the neurotrophic factor NT-3. Dendritic cell subsets secreting neurotrophic factor NT-3 are in addition to NT-3, immature dendritic cell subsets expressing CNTF, TGF-β1, IL-6, or NT-3, and CNTF The therapeutic agent for nerve injury or neurological dysfunction according to claim 8, which is a subset of mature dendritic cells expressing TGF-β1, IL-6, and EGF. The dendritic cell subset secreting neurotrophic factor NT-3 is an immature dendritic cell subset having a surface marker of CD11c on the cell surface or a mature dendritic cell subset derived from the immature dendritic cell. The nerve damage or nerve dysfunction disease therapeutic agent according to claim 8 or 9, characterized in. The mature dendritic cell subset is a mature dendritic cell subset obtained by culturing an immature dendritic cell subset in vitro in the presence of a stimulant to mature the immature dendritic cells. The therapeutic agent for nerve injury or neurological dysfunction according to claim 9 or 10. 12. The nerve according to any one of claims 9 to 11, wherein the mature dendritic cell subset is a mature dendritic cell subset into which an expression system for a protein or peptide of a nervous system or a gene encoding them is introduced. Drugs for damage or neurological dysfunction. A therapeutic agent for nerve damage or nerve dysfunction disease according to any one of claims 1 to 12, wherein the therapeutic agent for nerve damage or nerve dysfunction disease is administered to a nerve damage site, subcutaneously or in the vicinity of a lymph node, or intravenously. Method of treatment.

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