JP5198433B2 - 組織保存液の評価方法 - Google Patents
組織保存液の評価方法 Download PDFInfo
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- JP5198433B2 JP5198433B2 JP2009508930A JP2009508930A JP5198433B2 JP 5198433 B2 JP5198433 B2 JP 5198433B2 JP 2009508930 A JP2009508930 A JP 2009508930A JP 2009508930 A JP2009508930 A JP 2009508930A JP 5198433 B2 JP5198433 B2 JP 5198433B2
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Classifications
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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Description
非特許文献6には、GFPトランスジェニックラット由来の細胞が死滅した後においても、GFPが強い蛍光を発することが開示されている。
Science, vol.300(5616), p.87, 2003 Nat. Med., vol.4(2), p.245, 1998 Annu. Rev. Biomed. Eng., vol.4, p.235, 2002 Biochem. Biophys. Res. Commun., vol.329(1), p.288, 2005 Transplantation, vol.81, No.8, p.1179-1184, 2006 J. Biomed. Opt., vol.10(4), p.41204, 2005
[1]発光又は蛍光標識遺伝子が導入された哺乳動物組織を組織保存液に浸漬すること、浸漬後の該組織中の該標識遺伝子による発光又は蛍光レベルを測定すること、及び発光又は蛍光レベルに基づき組織保存液の保存効果を判定することを含む、組織保存液の保存効果の評価方法。
[2]該標識遺伝子がルシフェラーゼである、[1]記載の方法。
[3]該組織中の発光又は蛍光レベルが非破壊的に測定される、[1]記載の方法。
[4]該組織が発光又は蛍光標識遺伝子が導入された非ヒト哺乳動物から摘出されたものである、[1]記載の方法。
[5]該標識遺伝子が該哺乳動物において偏在性に発現している、[4]記載の方法。
[6]該標的遺伝子が該哺乳動物の目的組織において特異的に発現している、[4]記載の方法。
[7]組織保存液が細胞保存液である、[1]〜[6]のいずれか記載の方法。
[8]組織保存液が臓器保存液である、[1]〜[6]のいずれか記載の方法。
[9]哺乳動物組織として臓器の一部が用いられる、[1]記載の方法。
[10]以下の工程を含む、保存効果が確認された組織保存液の製造方法:
(I)所望の組織保存液の構成成分を配合し、該組織保存液を得ること;
(II)(I)で得られた組織保存液の一部をサンプルとして分離すること;
(III)発光又は蛍光標識遺伝子が導入された哺乳動物組織を(II)で分離されたサンプルに浸漬すること;
(IV)浸漬後の該組織中の該標識遺伝子による発光又は蛍光レベルを測定すること;
(V)発光又は蛍光レベルに基づきサンプルの保存効果を判定すること;及び
(VI)(V)において所望の保存効果が確認されたサンプルが由来する組織保存液を、保存効果が確認された組織保存液として得ること。
(I)所望の組織保存液の構成成分を配合し、該組織保存液を得ること;
(II)(I)で得られた組織保存液の一部をサンプルとして分離すること;
(III)発光又は蛍光標識遺伝子が導入された哺乳動物組織を(II)で分離されたサンプルに浸漬すること;
(IV)浸漬後の該組織中の該標識遺伝子による発光又は蛍光レベルを測定すること;
(V)発光又は蛍光レベルに基づきサンプルの保存効果を判定すること;及び
(VI)(V)において所望の保存効果が確認されたサンプルが由来する組織保存液を、保存効果が確認された組織保存液として得ること。
試験方法
試験溶液(組織保存液又は生理食塩水)にルシフェリンを適量溶解し、発光用溶液とした。発光用溶液中のルシフェリン濃度は150μg/mlであった。組織保存液としては、ET-Kyoto液を用いた。ルシフェラーゼ遺伝子を導入したLewisラット(Luc-LEW)(Transplantation, vol.81, No.8, p.1179-1184, 2006)から臓器(心臓、肺、腎臓、腸、膵臓、脾臓、肝臓)を取り出し、臓器中の血液を取り除いた後、該臓器を4℃の発光用溶液中に浸けた。Luc-LEWラットは、ルシフェラーゼ遺伝子を偏在性に発現している(Transplantation, vol.81, No.8, 2006)。臓器を浸けてから5分後にリアルタイムin vivoイメージングシステム(IVIS Imaging System、Xenogen社)を用いて臓器からの発光光量を測定した。発光光量の測定の間、発光用溶液の温度は4℃に維持した。発光光量を測定後、そのまま冷蔵庫(4℃)中で臓器を保存した。24、48及び72時間後に発光用溶液を新しいものに交換し、交換から5分後に上記と同様にIVISを用いて臓器からの発光光量を測定した。発光光量を数値化し(単位:p/sec/cm2/sr)、試験溶液の臓器保存効果を比較した。
保存開始から0〜72時間後の臓器からの発光のイメージを図1〜4に、各臓器の発光光量の経時的変化を図5及び6に示す。
保存開始時においては、試験溶液としてET−Kyoto液を用いた場合の発光光量は、生理食塩水を用いた場合とほぼ同一であった。しかし、各臓器を生理食塩水中で72時間保存すると臓器からのルシフェラーゼ発光はほとんど認められなくなったが、ET−Kyoto液中で保存すると依然として臓器からの有意なルシフェラーゼ発光が確認された(図4〜6)。
以上の結果から、臓器からのルシフェラーゼ発光を指標として、組織保存液の保存効果を評価できることが示された。
ルシフェラーゼ遺伝子を導入したLewisラット(Luc-LEW) (Transplantation, vol. 81, No.8, p.1179-1184, 2006)から肝臓を取り出し、肝臓中の血液を取り除き、氷冷(4℃)で保護した。直ちに4℃の低温室内にて以下の作業を行った。組織スライサーにより摘出肝臓を直径3 mmにスライスした。肝臓スライスはそれぞれ約14 mgの均一なスライスとして作成された。試験溶液(組織保存液又は生理食塩水)220 μlを96wellプレートの各wellに注入し、検査する肝臓スライスを試験溶液に浸漬した。組織保存液としては、University of Wisconsin液、ET-Kyoto液、Euro-Collins液、Histidine-tryptophan ketoglutarate液又はPerfadex液を用いた。試験溶液中の最終的なルシフェリン濃度が190 μg/mlとなるように、インジェクターからルシフェリン溶液20 μlを添加し、プレートリーダー(Mithras LB 940、Berthold社)を用いて肝臓スライスからの発光光量を測定した。発光光量の測定後、試験溶液を新しいものと交換し、次の測定まで4℃で保存した。上記方法により、保存開始時(0時間)、及び保存開始から1、3及び6時間後の発光光量を測定し、試験溶液の臓器保存効果を比較した。
肝臓切片を生理食塩水中で保存すると、ルシフェリンの発光量は急激に減少したが、組織保存液を用いた場合には発光量の減少は緩やかであった。
以上の結果から、組織切片を使用しても、組織からのルシフェラーゼ発光を指標として、組織保存液の保存効果を評価できることが示された。
本発明は日本で出願された特願2007−064171(出願日:2007年3月13日)を基礎としており、その内容は本明細書に全て包含されるものである。
Claims (9)
- ルシフェラーゼ遺伝子が導入された哺乳動物組織を組織保存液に浸漬すること、浸漬後の該組織中のルシフェラーゼによる発光レベルを測定すること、及び発光レベルに基づき組織保存液の保存効果を判定することを含む、組織保存液の保存効果の評価方法。
- 該組織中の発光レベルが非破壊的に測定される、請求項1記載の方法。
- 該組織がルシフェラーゼ遺伝子が導入された非ヒト哺乳動物から摘出されたものである、請求項1記載の方法。
- ルシフェラーゼが該哺乳動物においてユビキタスに発現している、請求項3記載の方法。
- ルシフェラーゼが該哺乳動物の目的組織において特異的に発現している、請求項3記載の方法。
- 組織保存液が細胞保存液である、請求項1〜5のいずれか記載の方法。
- 組織保存液が臓器保存液である、請求項1〜5のいずれか記載の方法。
- 哺乳動物組織として臓器の一部が用いられる、請求項1記載の方法。
- 以下の工程を含む、保存効果が確認された組織保存液の製造方法:
(I)所望の組織保存液の構成成分を配合し、該組織保存液を得ること;
(II)(I)で得られた組織保存液の一部をサンプルとして分離すること;
(III)ルシフェラーゼ遺伝子が導入された哺乳動物組織を(II)で分離されたサンプルに浸漬すること;
(IV)浸漬後の該組織中のルシフェラーゼによる発光レベルを測定すること;
(V)発光レベルに基づきサンプルの保存効果を判定すること;及び
(VI)(V)において所望の保存効果が確認されたサンプルが由来する組織保存液を、保存効果が確認された組織保存液として得ること。
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JPH08325101A (ja) * | 1995-05-31 | 1996-12-10 | Seitai Kagaku Kenkyusho:Kk | 動物組織保存方法 |
WO2005115141A1 (ja) * | 2004-05-26 | 2005-12-08 | National University Corporation Kagawa University | 希少糖を利用した細胞・組織・臓器保存液及び該液を用いる保存方法 |
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