JP2019533025A - 免疫調節活性を有する均質多糖類及びその調製方法 - Google Patents
免疫調節活性を有する均質多糖類及びその調製方法 Download PDFInfo
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- JP2019533025A JP2019533025A JP2018518439A JP2018518439A JP2019533025A JP 2019533025 A JP2019533025 A JP 2019533025A JP 2018518439 A JP2018518439 A JP 2018518439A JP 2018518439 A JP2018518439 A JP 2018518439A JP 2019533025 A JP2019533025 A JP 2019533025A
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- polysaccharide
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- homogeneous polysaccharide
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
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Abstract
Description
ステップ1:水中で加熱し抽出し、アルコール沈殿を行い、全多糖類を調製する。具体的に、
猪苓生薬を粉砕し、300gをとり、そこに3 L脱イオン水を加え、室温で1時間浸漬後、100℃で加熱還流することで2回(1時間/回)抽出し、その後、ろ過し、濾液を併合して600 mlに濃縮した後、3000 rpmで10 min遠心分離し、次いで、エタノールの割合が80%を超えるように上清を調整し、4℃で一晩放置した後、上清を濾別し、沈殿して凍結乾燥させ、多糖類粗生成物を得、多糖類粗生成物に純水を加えて25〜35の溶液を作成する。
ステップ2:Sevag法により多糖類粗生成物のタンパク質を除去する。具体的には、
糖液、クロロホルム、n-ブタノールを25:4:1の比で添加し、分液ロートに投げて3min十分に振とうした後、静置分層し、有基層及び沈殿物を廃棄し、白色沈殿が発生しないまでタンパク質の除去を複数回繰り返し、その後、上清を取集し、ロータリーエバポレーターを用いて適切な体積に濃縮させた後、転移し、凍結乾燥させ、タンパク質を除去した多糖類粗生成物を得る。
ステップ3:DEAE-52セルロースで色素を除去する。具体的には、
タンパク質を除去した猪苓多糖類粗生成物にそれぞれ少量の水を加えて溶解させた後、前処理されたDEAE-52セルロースカラム(35×3cm)を通して脱色させ、フェノール-濃硫酸法により流出液に糖を検出できないまで、1mL/min超純水、0.1〜0.5%塩化ナトリウム溶液で溶出し、ピークに対応する溶出液を収集して、ロータリーエバポレーターで濃縮させた後、凍結乾燥、脱塩し、緩い白色粉末として純粋な多糖類を得る。
ステップ4:Sephadex G-100ゲルカラムにより純化する。
具体的には、純粋な多糖類を蒸留水に適量に溶解させ、Sephadex G-100ゲルカラムを通し、純水、0.1〜0.5%塩化ナトリウム溶液により流速1ml/minで溶出し、溶出液を10 mLごとに収集し、フェノール-濃硫酸法により常時監視し、検出管番号を横座標、吸光度を縦座標として多糖類溶出曲線を描き、ピークに対応する溶出液を収集し、凍結乾燥、脱塩し、均質多糖類を得る。
ステップ1:乾燥粉末であるセルロースを蒸留水に2〜5h浸漬し、不純物を除去した後、乾燥させる。
ステップ2: 0.5mol/LのHCL溶液で1〜3h浸漬し、pHが中性となるように脱イオン水で洗浄し、乾燥させる。
ステップ3:乾燥したセルロースを0.5mol/LのNaOH溶液に1〜3h浸漬し、pHが中性となるように脱イオン水で洗?し、乾燥させる。
調製された猪苓均質多糖類PPSを分離し、分子量及び単糖成分の測定を第三者検出機関に依頼する。図2に示すように、HPGPCゲル透過クロマトグラフィーにより測定した重量平均分子量は約6.88 kDaである。PPSをリフルオロ酢酸で加水分解し、無水酢酸でアセチル化した後、GC-MSにより分析した結果、その単糖成分はD-グルコースのみである。UV スペクトル分析において、190 nm での末端吸収は多糖類の典型的な吸収であり、260、280 nmに吸収ピークがないことがPPSに核酸及びタンパク質?を含まないことを示す。そのIRスペクトルには、多糖類の典型的な特徴吸収ピーク(3304.6,2926.9,1642.9,1362.9,1148.8,1077.8,1014.2,847.6 cm-1)を示す。IRスペクトルにもNH吸収ピークがないため、該多糖類にアミノ基又は結合タンパク質を含まないことを示す。PPSの赤外吸収スペクトルは、図2に示す赤外吸収スペクトルと基本的に一致する。
マウスマクロファージ細胞株RAW264.7をl00ml/Lウシ胎仔血清、1%の二重抗体を含むDMEMに接種し、37C、5% CO2のインキュベータで培養する。
ヒト膀胱移行上皮癌細胞系 T24 細胞を10 %ウシ胎仔血清、100 U/mlペニシリン-ストレプトマイシンを含むDMED培地で培養し、5% CO2、37 °Cのインキュベータに置く。対数増殖期のT24細胞を3.5×107の細胞/ウェルで培養皿で培養し、18mlの培養液を添加し、細胞を90%コンフルエントに増殖するまで48 h培養し、培養上清を収集して使用まで保存する。収集した培養上清を 0.22 μm のフィルタをろ過し、ろ過した上清培養液を-80°C冷蔵庫に凍結保存する。
マウスマクロファージ系 RAW264.7細胞を10%ウシ胎仔血清、100U/mlペニシリン-ストレプトマイシンを含むDMED培地に培養し、5% CO2、37°Cインキュベータに置く。対数増殖期のRAW264.7細胞とT24上清とを共培養する。培養液の40%はT24腫瘍細胞培養上清である。
RAW264.7細胞懸濁液を104細胞/ウェルの密度で96ウェルプレートに接種し、24h培養した後、液を除去し、それぞれシリーズ濃度が3.09625、7.8125、15.625、31.25、62.5、125、250、500、1000 μg/mLのPPSの培地に入れる。24 h培養し、各ウェルに20 μLの50 g? L-1 MTT溶液を加え、4 hインキュベートし続け、上清を捨て、各ウェルに200 μLのDMSOを加え、室温で溶解し、10 min振とうした後、マイクロプレートリーダーで490 nm波長の吸光度(OD)値を測定する。各薬物濃度について4ウェル繰り返し、細胞相対生存率を計算する。計算式:細胞相対生存率=OD490(投与)/OD490(対照)。OD490(投与)及びOD490(対照)は、それぞれ投与群細胞サンプル及び対照群液の細胞サンプルの、MTT実験における490nmでの吸収値である。PPSのRAW264.7マクロファージ生存率に対する影響を図5に示す。図5に示すように、薬物濃度が3.9-125 mg/mLである場合に、細胞生存率は、対照群と比較して統計的有意性がないため、PPS後続実験で選択される濃度勾配は1、10、100 mg/mLである。
増殖状態が良好なRAW264.7マクロファージを取り、消化して継代培養し、カウントし細胞密度を調整し、12ウェルの細胞培養プレートで培養し、1mlの細胞懸濁液を加え、各ウェルにRAW264.7細胞を2.5 ×105個を加え、37°C、5%CO2インキュベータに置いて24h培養した後、陽性群に最終濃度100ng/mLのIFN-γ溶液を加え、投与群にそれぞれ3.09625、7.8125、15.625、31.25、62.5、125、250、500、1000 μg/mLのPPSを加え24 h培養する。共培養群は、細胞を24h培養した後、40%の上清を取り除き、40%のT24上清夜を加え、3時間共培養し、その後、陽性群に最終濃度100ng/mLのIFN-γ溶液を与え、各投与群に3.09625、7.8125、15.625、31.25、62.5、125、250、500、1000 μg/mLのPPSを与えて24h培養する。細胞上清を収集し、Griess法によりNOサイトカインの分泌量を測定する。結果を図7Aに示す。結果として、PPSが3.9g/mlでIFN-γ(100ng/mL)よりも多いNOを分泌するように刺激し、PPSが膀胱癌シミュレーションの腫瘍微環境(マクロファージ-膀胱癌細胞共培養)においてマクロファージがNOを分泌することを顕著に促進でき、比較的な投与量3.90625μg/mLであるときに、NOの分泌量は、陽性対照群IFN-γ(100ng/mL)の2倍よりも多い。
マウスマクロファージ細胞株RAW264. 7を100ml/Lのウシ胎仔血清を含むDMEM培養液で培養する。対数増殖期にある細胞を選択し、消化して継代培養し、カウントし細胞密度を調整し、1×106個/ウェルで6ウェルの細胞培養プレートに接種する。完全に壁に付着するまで24 h培養する。0.8 mLの培地を取り除き、0.8 mLのT24上清を加え、3h培養する。それぞれ最終濃度が100ng/mLのIFN-γ溶液、最終濃度が1、10、100 μg/mLのPPSを与えて24h作用する。上清を取り除き、予冷したPBS緩衝液を加えて2回洗浄し、その後、適量のRIPAライセート(ホスファターゼ阻害剤及びタンパク質?阻害剤含有)を加え、氷で細胞を擦り取り、15000×gで15 min遠心分離し、細胞総タンパク質溶液として上清を取る。ローディングバッファー和及び超純水を加える(100°C、10min)。Western-blotデータ分析を行った結果、対照群と比較して、1、10、100 μg/mLのPPSは、いずれもCD14、CD284(TLR4)、CD282(TLR2)受容体発現を顕著に活性化し、P-P38、P-P65プロテインキナーゼリン酸化発現を増加する作用を有する(図9)。それによって、PPSが膀胱癌腫瘍微環境でマクロファージを活性化するのは、CD14/TRL4/P38 MAPK及びTRL2//NF-kB経路を活性化することで免疫調節作用を発揮することが示唆される。
Claims (6)
- 単一のクロマトグラフィーのピークを有し、分子量が6880±50Da、旋光度が158.4±0.5°であり、赤外吸収スペクトルにおいて847.6 cm-1に特徴的な吸収ピークを示し、水素スペクトルにおける水素プロトンの化学シフトがそれぞれδ 5.40(brs),3.95(t,J =7.2 Hz),3.84m,3.61mであり、炭素スペクトルにおける炭素信号の化学シフトがそれぞれδ99.6,δ76.6,δ73.3,δ71.5,δ71.1,δ60.4であることを特徴とする免疫調節活性を有する均質多糖類。
- 上記均質多糖類における多糖類の含有量(質量百分率)が92〜98%であり、Agilent1200液体クロマトグラフィー(検出器:示差屈折率検出器RID;カラム:TSK-GEL G4000 PWxL;移動相:超純水;流速:0.3〜1ml/min;検出器温度:30〜40℃;カラム温度:30〜50℃)により分析した結果、単一のクロマトグラフィーのピークを示し、保持時間が10〜30 minであることを特徴とする請求項1に記載の免疫調節活性を有する均質多糖類。
- 上記均質多糖類における単糖成分は、α-D-グルコピラノースであり、構造が全てα型であり、結合方式が1→4結合である請求項1に記載の免疫調節活性を有する均質多糖類。
- 前記均質多糖類に、蛍光標識生成物、カルボキシメチル化生成物、メチロール化生成物、ヒドロキシプロピル化生成物、エチレングリコール化生成物、プロピレングリコール生成物或ポリエチレングリコール化生成物のうちの1種がさらに添加されている請求項1に記載の免疫調節活性を有する均質多糖類。
- 請求項1に記載の免疫調節活性を有する均質多糖類の調製方法であって、該方法は、以下のステップ1〜4を含み、
ステップ1:水中で加熱し抽出し、アルコール沈殿を行い、全多糖類を調製し、具体的に、
猪苓生薬を粉砕し、300gをとり、そこに3 L脱イオン水を加え、室温で1時間浸漬後、100℃で加熱還流することで2回(1時間/回)抽出し、その後、ろ過し、濾液を併合して600 mlに濃縮した後、3000 rpmで10 min遠心分離し、次いで、エタノールの割合が80%を超えるように上清を調整し、4℃で一晩放置した後、上清を濾別し、沈殿して凍結乾燥させ、多糖類粗生成物を得、多糖類粗生成物に純水を加えて25〜35の溶液を作成し、
ステップ2:Sevag法により多糖類粗生成物のタンパク質を除去し、具体的には、
糖液、クロロホルム、n-ブタノールを25:4:1の比で添加し、分液ロートに投げて3min十分に振とうした後、静置分層し、有基層及び沈殿物を廃棄し、白色沈殿が発生しないまでタンパク質の除去を複数回繰り返し、その後、上清を取集し、ロータリーエバポレーターを用いて適切な体積に濃縮させた後、転移し、凍結乾燥させ、タンパク質を除去した多糖類粗生成物を得て、
ステップ3:DEAE-52セルロースで色素を除去し、具体的には、
タンパク質を除去した猪苓多糖類粗生成物にそれぞれ少量の水を加えて溶解させた後、前処理されたDEAE-52セルロースカラム(35×3cm)を通して脱色させ、フェノール-濃硫酸法により流出液に糖を検出できないまで、1mL/min超純水、0.1〜0.5%塩化ナトリウム溶液で溶出し、ピークに対応する溶出液を収集して、ロータリーエバポレーターで濃縮させた後、凍結乾燥、脱塩し、緩い白色粉末として純粋な多糖類を得て、
ステップ4:Sephadex G-100ゲルカラムにより純化し、具体的には、
純粋な多糖類を蒸留水に適量に溶解させ、Sephadex G-100ゲルカラムを通し、純水、0.1〜0.5%塩化ナトリウム溶液により流速1ml/minで溶出し、溶出液を10 mLごとに収集し、フェノール-濃硫酸法により常時監視し、検出管番号を横座標、吸光度を縦座標として多糖類溶出曲線を描き、ピークに対応する溶出液を収集し、凍結乾燥、脱塩し、均質多糖類を得る、免疫調節活性を有する均質多糖類の調製方法。 - 上記ステップ3におけるDEAE-52セルロースカラムの前処理方法は以下のステップ1〜3を含み、
ステップ1:乾燥粉末であるセルロースを蒸留水に2〜5h浸漬し、不純物を除去した後、乾燥させ、
ステップ2: 0.5mol/LのHCL溶液で1〜3h浸漬し、pHが中性となるように脱イオン水で洗浄し、乾燥させ、
ステップ3:乾燥したセルロースを0.5mol/LのNaOH溶液に1〜3h浸漬し、pHが中性となるように脱イオン水で洗?し、乾燥させる、請求項5に記載の免疫調節活性を有する均質多糖類の調製方法。
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AU2018202402B2 (en) | 2020-02-06 |
JP6681466B2 (ja) | 2020-04-15 |
CN107698695B (zh) | 2021-06-01 |
EP3480218A1 (en) | 2019-05-08 |
CN107698695A (zh) | 2018-02-16 |
EP3480218A4 (en) | 2020-02-12 |
AU2018202402A1 (en) | 2019-03-28 |
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