JP6136151B2 - 抗疲労物質の評価方法 - Google Patents
抗疲労物質の評価方法 Download PDFInfo
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Description
(1)生体試料中のリボヌクレアーゼL又はそのホモログの発現量を指標とした被験物質の疲労に対する有効性を判定する方法。
(2)生体試料が、血液又は肝臓又は筋肉である前記(1)に記載の被験物質の疲労に対する有効性を判定する方法。
(3)疲労が、肉体疲労である前記(1)から(2)記載の被験物質の疲労に対する有効性を判定する方法。
試験例1 抗疲労物質の作用評価
トレッドミル装置(Panlab社製)の傾斜角を10°に設定し、BALB/c雄性マウス(日本エスエルシー(株)、1群8匹)に、漸増式(15cm/sから4分毎に5cm/sずつ速度を増加)に走行運動を負荷し、走行不能となる第1限界走行速度を算出した。その後、休憩時間を4時間挟み、再度、同じ条件にて、走行運動を負荷し、走行不能となる第2限界走行速度を算出した。
被験物質(タウリン)は走行運動試験の前日まで1日1回で21日間の連続投与を行った(300mg/kg、p.o)。走行運動試験当日は、1度目の走行運動試験終了直後に投与した。
行った試験は、(1)走行運動の負荷、(2)組織の採取、(3)RNAの抽出、(4)遺伝子の発現量の分析及び比較、の工程よりなる。尚試験には、被験物質のタウリンを投与したタウリン群とその溶媒を投与した対照群を用いた。
(1)走行運動の負荷
トレッドミル装置(Panlab社製)の傾斜角を10°に設定し、BALB/c雄性マウス(日本エスエルシー(株)、1群4匹)に、漸増式(15cm/sから4分毎に5cm/sずつ速度を増加)に走行運動をさせ、疲労を負荷した。タウリンおよび溶媒は走行運動試験の前日まで1日1回で21日間の連続投与を行った(300mg/kg、p.o)。
(2)組織の採取
走行運動負荷群は、走行4時間後に深麻酔条件下で腹部大静脈から血液を1mL採取した。採取した血液には、ヌクレアーゼ不含水1mLを添加し、Isogen−LS(ニッポンジーン)2mLを添加し、血液を溶解した。溶解した血液は液体窒素中で急速凍結後、−80℃で一時保管し、後日RNA抽出に供した。非走行群に対しても、同様の方法で採取した。
(3)RNAの抽出
Isogen−LS(ニッポンジーン社)に溶解されている血液サンプル4mLの半量を用いて精製を行った。Isogen−LS溶解サンプル2mLに対し、クロロホルム400μLを加えて、十分ボルテックスし遠心の後、水層800から1000μLをRNA粗抽出液として回収した。得られたRNA粗抽出液をRNeasy Mini kit中のColumnに流し入れ、RNAを吸着させた。洗浄後60μLのヌクレアーゼ不含水に溶解させた。RNAを含むヌクレアーゼ不含水1μLを、NanoDropSpectrophotometer(NanoDrop社)を用いてRNAの定量及び純度指標による品質確認を行った。
Affymetrix社において作製されたゲノム上の既知、及び想定される28853個の遺伝子について25mer×770317個の対象と完全一致するオリゴヌクレオチド・プローブ(パーフェクトマッチプローブ)が搭載されたDNAマイクロアレイ(GeneChip Mouse Gene 1.0 ST Array)を用い、遺伝子発現量の解析を行った。具体的には、次の手順で行った。
WT Expression Kit(Ambion社)を用い、プロトコールに従って、250ngのTotal RNAより、anti−sense鎖cRNAの合成を介して、sense 鎖cDNAを合成した。その後、WT Terminal Labeling and Control Kit(Affymetrix社)を用いて、合成したcDNAを断片化し、その5’末端にビオチンラベルを行った。中途産物であるcRNAを常法により変性後、断片化前のcDNA及び断片化後のcDNA(100から375ng)をBioAnalyzer 2100を用いて電気泳動し、品質確認を行った。
Claims (2)
- 生体試料中の配列番号1で規定されるリボヌクレアーゼL遺伝子の塩基配列と95%以上の同一性を有し、且つリボヌクレアーゼ活性を有するタンパク質をコードする遺伝子、又は配列番号1で規定される該遺伝子によりコードされるタンパク質のアミノ酸配列と95%以上の同一性を有し、リボヌクレアーゼ活性を有するタンパク質の発現量が増加することを指標とした被験物質の肉体疲労に対する有効性を判定する方法。
- 生体試料が、血液又は肝臓又は筋肉である請求項1に記載の被験物質の肉体疲労に対する有効性を判定する方法。
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Publication number | Priority date | Publication date | Assignee | Title |
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US5985565A (en) * | 1996-10-09 | 1999-11-16 | Temple University-Of The Commonwealth System Of Higher Education | Chronic fatigue syndrome diagnosis |
JP2004251753A (ja) * | 2003-02-20 | 2004-09-09 | Taisho Pharmaceut Co Ltd | 疲労回復作用の評価方法 |
US20070134156A1 (en) * | 2004-02-17 | 2007-06-14 | Soiken Inc. | Fatigue evaluation apparatus, fatigue evaluation method, and application thereof |
JP2007114170A (ja) * | 2005-10-18 | 2007-05-10 | Sogo Ikagaku Kenkyusho:Kk | 疲労度評価方法およびその利用 |
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