JP2011506418A - 眼における細胞死についての蛍光マーカー - Google Patents
眼における細胞死についての蛍光マーカー Download PDFInfo
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Abstract
Description
組換えにより細菌で発現させたアネキシン5(等張緩衝液1ml中5mg)が、「標識化緩衝液」に対し、緩衝液を2回交換して(各々250ml)3時間かけて透析される。標識化緩衝液は、炭酸水素ナトリウム21g(250mmol)を蒸留水400mlに溶解することによって作製される。アジ化ナトリウム1g(0.2%)を添加する。水酸化ナトリウムの濃厚水溶液でpHを9.0に調整する。使用の前に、濃縮保存溶液1容に対し水9容(v/v)を添加して緩衝液を希釈する。アネキシン5タンパク質は、今や標識化反応の準備ができている。染料対タンパク質比は、各々の標識化反応について一定に保持される。それ故、アネキシン5 1mgを標識するためにD776−NHS−エステル0.5mgを使用する。これは、染料とアネキシン5タンパク質の間で約500のモル比を与える。NHS−エステルをボルテックスすることによってジメチルホルムアミドに溶解し、染料を標識化緩衝液中のアネキシン5タンパク質に添加して反応を開始させる。標識化反応におけるアネキシン5の最終濃度は5mg/mlであるべきである。標識化は、振とう機に入れたエッペンドルフ管において2時間にわたって進行させることが可能である。
画像化は、当分野で公知の手法、例えば以下のような記述されている手法を用いて実施し得る。
本発明者らは、網膜における内因性蛍光シグナルの存在は、特定の蛍光標識で標識された細胞死マーカーが適切に撮像できないことを意味することを発見した。本発明者らは以前に、網膜神経節細胞のアポトーシスを撮像するために蛍光標識アネキシン(アネキシン488)を使用した。全身的に投与した場合、本発明者らは、驚くべきことに、固有の網膜自己蛍光が、有用な結果が得られない程度までシグナルに干渉することを発見した。
トランスジェニックモデルにおける糖尿病性神経変性のDARC評価を、本明細書で述べた手法を用いて実施した。図5に見られるように、糖尿病動物(a)は、年齢適合野生型対照(b)よりも有意に多い網膜神経節細胞アポトーシスを示す。
標識細胞におけるインビボ対組織学の相違
a)硝子体内投与
本発明者らは以前に、網膜神経節細胞のアポトーシスを画像化するために、硝子体内経路で眼に投与した蛍光標識アネキシン(アネキシン488)を使用した。Alexa Fluora 488標識アネキシン5又は別の蛍光マーカーで標識したアネキシン5。それらは以下のように評価された。
・アネキシンAlexa Flour 532 532/553の最大励起/蛍光波長
・アネキシンAlexa Fluor 555 555/568の最大励起/蛍光波長
・アネキシンDy−776 771/793の最大励起/蛍光波長
・アネキシンDy−781 783/800の最大励起/蛍光波長
・アネキシンLicor IRDye 800 774/789の最大励起/蛍光波長。
本発明者らは、他のアネキシンマーカー、例えば、すべてが暴露ホスファチジルセリンに対して親和性を有する、アネキシン11、アネキシン2及びアネキシン6も、アポトーシスを起こしている網膜細胞に結合することを示した。
ヨウ化プロピジウム
本発明者らは、次に細胞死の他のマーカーを評価した。最初に、分断された膜を有する細胞を標識し、それらのDNA及びRNAを蛍光染色することによって壊死細胞を同定するヨウ化プロピジウム(PI)。PIは、それぞれ532nmと649nmの最大励起及び蛍光波長を有する。図19の画像は、網膜においてインビボで壊死細胞を標識するPIとインビボでアポトーシス細胞を標識するアネキシン488の比較を示す。
メロシアニン540は、細胞の外膜に結合し、アポトーシスの初期に起こる膜の解体の程度に比例して蛍光を発するという事実によってアポトーシス細胞を同定する染料である。MC450は、それぞれ488nmと575nmの最大励起及び蛍光波長を有する。図20の画像は、食塩水とSSPで処置した眼のインビボでのMC540標識の比較、及びアネキシン776標識との比較を示す。
本発明者らは以前に、Anx−FがRGCアポトーシスを同定するために使用し得ることを示した。最近、同じ手法を使用して、アポトーシスを起こしている双極細胞を視覚化することが可能であることを示した。本発明者らはここで、図21に見られるように、硝子体内投与した場合のAnx−Fが他の網膜細胞も染色できることを示す。
Claims (22)
- 眼における細胞死を同定するための、波長最適化標識で標識した細胞死マーカーの使用。
- 前記細胞死マーカーがアネキシン又はそのフラグメント若しくはその誘導体である、請求項1に記載の使用。
- 前記細胞死マーカーがアネキシン5、11、2又は6である、請求項2に記載の方法。
- 前記波長最適化標識がD−776である、請求項1から3のいずれか一項に記載の使用。
- 前記マーカーが網膜細胞死を同定するためである、請求項1から4のいずれか一項に記載の使用。
- 前記マーカーが網膜神経細胞死を同定するためである、請求項5に記載の使用。
- 前記マーカーが単一細胞の死を同定するためである、請求項1から6のいずれか一項に記載の使用。
- 眼における細胞死を観測するための方法であって、
(a)波長最適化標識で標識した細胞死マーカーを被験者に投与するステップと、
(b)画像化装置を使用して被験者の眼から励起波長の画像を作製するステップとを含み、前記画像が眼における細胞死の表示である方法。 - 前記細胞死マーカーがアネキシンである、請求項8に記載の方法。
- 前記細胞死マーカーがアネキシン5である、請求項9に記載の方法。
- 前記波長最適化標識がD−776である、請求項1から10のいずれか一項に記載の方法。
- 前記マーカーが網膜細胞死を同定するためである、請求項8から11のいずれか一項に記載の方法。
- 前記マーカーが網膜神経細胞死を同定するためである、請求項12に記載の方法。
- 前記マーカーが単一細胞の死を同定するためである、請求項8から13のいずれか一項に記載の方法。
- 前記標識マーカーが全身的、局所的又は経眼的に投与される、請求項8から14のいずれか一項に記載の方法。
- 画像を作製する前記ステップを反復する、請求項8から15のいずれか一項に記載の方法。
- 画像を作製する前記ステップを、リアルタイム画像化を可能にするように反復する、請求項16に記載の方法。
- 前記方法が前記眼の変性疾患を診断するためである、請求項8から17のいずれか一項に記載の方法。
- 前記疾患が緑内障又は糖尿病性網膜症である、請求項18に記載の方法。
- 前記疾患が脳の神経変性疾患である、請求項8から17のいずれか一項に記載の方法。
- 前記疾患がアルツハイマー病である、請求項20に記載の方法。
- 最適化波長標識で標識した細胞死マーカーを含有する医薬組成物であって、静脈内、経眼又は局所投与に適する医薬組成物。
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PCT/GB2008/004156 WO2009077750A1 (en) | 2007-12-14 | 2008-12-15 | Fluorescent marker for cell death in the eye |
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JP2011051961A (ja) * | 2009-09-04 | 2011-03-17 | Mie Univ | 近赤外光発光化合物、その合成法及びその発光法 |
JP2021522215A (ja) * | 2018-04-23 | 2021-08-30 | ユーシーエル ビジネス リミテッド | 蛍光マーカーの鼻腔内送達 |
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GB0919520D0 (en) | 2009-11-06 | 2009-12-23 | Ucl Business Plc | Quantifying cell death |
RU2657515C2 (ru) * | 2010-04-13 | 2018-06-14 | м-лаб ГмбХ | Способы диагностики глаукомы |
NL2008241C2 (en) * | 2012-02-06 | 2013-08-08 | Hq Medical Netherlands B V | Cell death probe. |
KR20160021789A (ko) * | 2013-05-23 | 2016-02-26 | 뉴로슨, 인크. | 방사성 핵종으로 분류된 아넥신을 사용해서 알츠하이머 질환의 진단 및 치료 |
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