JP7461034B2 - 間葉系幹細胞増殖剤 - Google Patents
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Description
1)細胞
・脂肪由来幹細胞(PT-5006、Lonza、Batch No:0000605220、Tissue Acquisition Number:31363)
2)ローヤルゼリー
・未酵素分解ローヤルゼリー(山田養蜂場社。生ローヤルゼリーを乾燥、粉末化したもの。)
・酵素分解ローヤルゼリー(山田養蜂場社)
酵素分解ローヤルゼリーとしては、pH7.8に調整した未酵素分解ローヤルゼリー水溶液をプロテアックス(天野エンザイム株式会社)で50℃、2時間反応させることにより加水分解したものを用いた。
3)培地、試薬、器材等
・増殖用培地:間葉系幹細胞用無血清培地(FKCM 301T、フコク)
・FBS(ウシ胎児血清、BI、04-400-1A)
・IMDM(イスコブ変法ダルベッコ培地、ナカライテスク、11506-05)
・生細胞数測定試薬SF (ナカライテスク、07553-44)
・シリンジフィルター(6896-2502、Whatman-GE)
未酵素分解ローヤルゼリー粉末又は酵素分解ローヤルゼリー粉末を精製水に懸濁、撹拌し、5分間超音波破砕したものをそれぞれ未酵素分解ローヤルゼリー原液又は酵素分解ローヤルゼリー原液とした。培地9mlに、未酵素分解ローヤルゼリー原液又は酵素分解ローヤルゼリー原液1mlを添加してボルテックスで撹拌し、10分間室温で放置した後、0.2μmフィルターでろ過滅菌することにより各ローヤルゼリーの10%溶液を調製した。
脂肪由来幹細胞を上述の増殖培地で1×105cells/mLに調製し、96ウェルプレートに100μL/wellで播種した。翌日に95~100%コンフルエントの状態になった細胞を細胞毒性試験に用いた。ローヤルゼリーサンプルは増殖培地で1%濃度になるように調製し、更に同培地で2倍希釈して、0.004%までの9段階の濃度を作製した。ウェルの培地を除去して、調製したローヤルゼリーサンプルを添加して37℃のCO2インキュベーターで24時間培養した。24時間後に培地を除去し、生細胞数測定試薬SFを10%添加したIMDMを75μL/wellで添加し、1~2時間インキュベートして450nmの吸光度を測定した。細胞毒性試験は、1濃度につき3wellで行い、ブランクの吸光度を100%として各濃度の細胞生存率(%)を算出した。
脂肪由来幹細胞をIMDMで3×104cells/mLに調製し、96ウェルプレートに100μL/wellで播種し、翌日に30%コンフルエントの状態になった細胞を増殖促進試験に用いた。ローヤルゼリーサンプルとしては、増殖培地(2%FBS増殖添加増殖培地)、及び増殖培地をIMDMで10倍希釈した培地(0.2%FBS増殖添加増殖培地)の2種類で、それぞれ2倍希釈系列のサンプルを調製した。ウェルの培地を除去して、調製したローヤルゼリーサンプルを添加して37℃のCO2インキュベーターで3日間培養した。培地を除去し、生細胞数測定試薬SFを10%添加したIMDMを75μL/wellで添加し、1~2時間インキュベートした。その後450nmの吸光度を測定した。測定は1濃度につき3wellで行った。結果を図1及び図2に示す。
Claims (2)
- ローヤルゼリーを有効成分として含み、哺乳類動物の胎盤又は植物種属の胎座から抽出された胎盤抽出物を含まない、間葉系幹細胞増殖剤。
- 前記ローヤルゼリーが酵素分解ローヤルゼリーである、請求項1に記載の剤。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2002112715A (ja) | 2000-10-03 | 2002-04-16 | Api Co Ltd | 低アレルゲン化ローヤルゼリー及びその製造方法 |
JP2007295919A (ja) | 2006-04-06 | 2007-11-15 | Yamada Bee Farm Corp | 低アレルゲン化ローヤルゼリーの製造方法 |
JP2016113442A (ja) | 2014-12-11 | 2016-06-23 | 國璽幹細胞應用技術股▲分▼有限公司 | 不老回春栄養補給剤 |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2002112715A (ja) | 2000-10-03 | 2002-04-16 | Api Co Ltd | 低アレルゲン化ローヤルゼリー及びその製造方法 |
JP2007295919A (ja) | 2006-04-06 | 2007-11-15 | Yamada Bee Farm Corp | 低アレルゲン化ローヤルゼリーの製造方法 |
JP2016113442A (ja) | 2014-12-11 | 2016-06-23 | 國璽幹細胞應用技術股▲分▼有限公司 | 不老回春栄養補給剤 |
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