JP2023510291A - 幹細胞の機能強化用組成物 - Google Patents
幹細胞の機能強化用組成物 Download PDFInfo
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Abstract
Description
本発明の様態によれば、IL-1β及びビタミンB6;またはIL-1β、ビタミンB6及びIFN-αを含む幹細胞の免疫調節及び炎症調節能増進用組成物を提供する。
37℃及び5%CO2インキュベーターで、貯蔵状態(LN2 タンク保管)の中間葉幹細胞を解凍して培養し、このとき、10%FBSまたは4%hPLを含有する培地(DMEM、α-MEM)で細胞コンフルエンスが80%程度に増殖するまで培養した。培養した中間葉幹細胞を100mmディッシュに播種した後、中間葉幹細胞機能強化のための候補物質を選定した。
1.1 実験群処理によるIFN-γの分泌の確認
前記表1のような実験群処理によって中間葉幹細胞の活性が変化したかを確認するために、免疫調節活性物質であるPHA(Phytohemagglutinin)刺激条件でT-細胞を活性化させた後、中間葉幹細胞を3日間共同培養した。この後、共同培養した上清液を収得してIFN-γ kit(BD Biosciences)を通して前炎症性サイトカインであるIFN-γの分泌を確認して中間葉幹細胞の免疫調節能変化の有無を確認した。対照群としては、実験物質を処理していない中間葉幹細胞を利用した。実験群処理によるIFN-γの分泌を図3に示した。
ICOSL(誘導性共刺激リガンド)は、調節T細胞を誘導させて活性化されたT細胞を抑制するものと知られている。実施例1.1において実験群処理による中間葉幹細胞の機能強化効果を確認したので、実験群処理が中間葉幹細胞の炎症及び免疫調節能力を誘導するかをさらに確認するための実験を遂行した。実験例1のように幹細胞を培養した後、TRIzol(Invitrogen)を利用して総RNAを分離した後、PrimeScript(商標)reagent Kit with gDNA Eraser(TaKaRa)を利用して総RNAでcDNAを合成してqRT-PCRを遂行した。これを通して炎症及び免疫調節の代表的な因子であるICOSLの発現変化を確認し、その結果を図4に示した。
ガレクチン1は、幹細胞高効能と密接な関連のある幹細胞効能指標(potency marker)と知られており、発現が増加するほど幹細胞の機能及び効能が強化されるものと知られている。従って、実験群処理が中間葉幹細胞の炎症及び免疫調節能力を誘導するかをさらに確認するためにガレクチン1発現確認実験を遂行した。実験例1のように幹細胞を培養した後、TRIzol(Invitrogen)を利用して総RNAを分離した後、PrimeScript(商標)reagent Kit with gDNA Eraser(TaKaRa)を利用して総RNAでcDNAを合成してqRT-PCRを遂行した。これを利用して幹細胞効能指標であるガレクチン1の発現変化を確認し、その結果を図5に示した。
前記実施例1.2及び1.3を通してIL-1βとビタミンB6の組み合わせ、またはIL-1β、IFN-α及びビタミンB6の組み合わせが免疫及び炎症調節のための中間葉幹細胞の機能を強化できることを確認したので、最も優れた効果を示すIL-1β、IFN-α及びビタミンB6の組み合わせに対して追加実験を遂行した。IL-1β、IFN-α及びビタミンB6の組み合わせを中間葉幹細胞に処理し、前記実施例1.2と同じ実験方法でIDO発現変化誘導効果を確認し、その結果を図6に示した。
Claims (15)
- IL-1β及びビタミンB6を含む、幹細胞の免疫調節及び炎症調節能増進用組成物。
- IFN-αをさらに含む、請求項1に記載の幹細胞の免疫調節及び炎症調節能増進用組成物。
- 前記IL-1β及びビタミンB6は、1:1~1:6,000(w/v)の比率で含まれるものである、請求項1に記載の幹細胞の免疫調節及び炎症調節能増進用組成物。
- 前記IL-1β、IFN-α及びビタミンB6は、1:0.01~5:1~6,000(w/v)の比率で含まれるものである、請求項2に記載の幹細胞の免疫調節及び炎症調節能増進用組成物。
- 前記幹細胞は、脂肪、骨髄、胎盤または臍帯血由来中間葉幹細胞である、請求項1乃至4のいずれか一項に記載の幹細胞の免疫調節及び炎症調節能増進用組成物。
- 前記IL-1β、ビタミンB6;またはIL-1β、IFN-α及びビタミンB6;は、IFN-γ抑制増進、ICOSL(誘導性共刺激リガンド)発現増加、IDO(インドールアミン-2,3-ジオキシゲナーゼ)発現増加及びガレクチン1発現増加からなる群から選択された1種以上の幹細胞の免疫調節及び炎症調節能増進を誘導するものである、請求項1または2に記載の幹細胞の免疫調節及び炎症調節能増進用組成物。
- 1)IL-1β及びビタミンB6を幹細胞に処理して培養するステップ;を含む、幹細胞の免疫調節及び炎症調節能増進方法。
- 前記1)ステップでIFN-αを共に処理するものである、請求項7に記載の幹細胞の免疫調節及び炎症調節能増進方法。
- 前記幹細胞の免疫調節及び炎症調節能増進は、IFN-γ抑制増進、ICOSL(誘導性共刺激リガンド)発現増加、IDO(インドールアミン-2,3-ジオキシゲナーゼ)発現増加及びガレクチン1発現増加からなる群から選択された1種以上である、請求項7に記載の幹細胞の免疫調節及び炎症調節能増進方法。
- 1)IL-1β及びビタミンB6を幹細胞に処理して培養するステップ;を含む、免疫調節及び炎症調節能が増進された幹細胞の製造方法。
- 前記1)ステップでIFN-αを共に処理するものである、請求項10に記載の免疫調節及び炎症調節能が増進された幹細胞の製造方法。
- 請求項10または11に記載の方法で製造された免疫調節及び炎症調節能が増進された幹細胞。
- 請求項12に記載の幹細胞を含む、免疫疾患予防または治療用薬学的組成物。
- 前記免疫疾患は、クローン病、紅斑病、アトピー、関節リウマチ、橋本甲状腺炎、悪性貧血、アジソン病、1型糖尿病、全身性エリテマトーデス、慢性疲労症候群、線維筋痛症、甲状腺機能低下症、亢進症、強皮症、ベーチェット病、炎症性腸疾患、多発性硬化症、重症筋無力症、メニエール症候群(Meniere’s syndrome)、ギラン・バレー症候群(Guillain-Barre syndrome)、シェーグレン症候群(Sjogren’s syndrome)、白斑症、子宮内膜症、乾癬、全身性強皮症、喘息及び潰瘍性大腸炎からなる群から選択された1種以上であることを特徴とする、請求項13に記載の免疫疾患予防または治療用薬学的組成物。
- 請求項12に記載の幹細胞を個体に処理するステップ;を含む、免疫疾患予防または治療方法。
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