JP6822412B2 - 妊娠高血圧腎症のための診断アッセイ及び治療 - Google Patents
妊娠高血圧腎症のための診断アッセイ及び治療 Download PDFInfo
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- JP6822412B2 JP6822412B2 JP2017543799A JP2017543799A JP6822412B2 JP 6822412 B2 JP6822412 B2 JP 6822412B2 JP 2017543799 A JP2017543799 A JP 2017543799A JP 2017543799 A JP2017543799 A JP 2017543799A JP 6822412 B2 JP6822412 B2 JP 6822412B2
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Description
典型的には、血中のsFlt−1:PlGFと尿素との間の正常な比は、5.2未満である。
典型的には、血中のsFlt−1:尿素の正常な比は、1020未満である。
典型的には、血中のsFlt−1とビリルビン:尿素との間の正常な比は、698.4未満である。
典型的には、血中のPlGFとビリルビン:尿素との間の正常な比は、161.4超である。
典型的には、血中のPlGFとビリルビンとの間の正常な比は、33.94超である。
典型的には、血中のPlGFと尿素との間の正常な比は、45.09超である。
(i)シトルリンと、ASl及びASSの活性化剤と、又は
(ii)アルギニノコハク酸と、ASSの活性化剤と
の組合せを使用する、方法も提供する。これらは、図10に示す経路の構成成分であり、これらにより、妊娠高血圧腎症を治療することが可能になることが予測される。
患者の試料
予測研究:これは、前向き研究であり、2012年9月〜2014年12月の間に、St George’s Hospital University of Londonにて行われた。上記研究に参加することを合意する全て女性から、書面によるインフォームドコンセントを得た。上記研究は、London−Stanmore Research倫理委員会により承認された。この研究についての組入れ基準は、20+0週の妊娠以降に妊娠高血圧腎症が疑われる症状又は徴候を示す、単生児妊娠を有する女性であった。アッセイのオペレーターは、参加者の臨床情報について知らされなかった。母体の血液を、静脈穿刺により、抗凝固薬を用いずに収集した。試料を、2000×gで遠心分離し、血清を、ピペット採取し、試験するまで−80℃で保存した。Roche Diagnostics、Penzberg、ドイツ製のエレクシス(Elecsys)プラットフォーム上の市販されているアッセイにより、sFlt−1及びPlGFの濃度を、平行して決定し、次いで、測定した。母体の血清ビリルビンレベルを、比色分析アッセイキットを使用して測定した。
妊娠していないマウスを用いる研究を、12週齢のHmox1−/−マウス及びHmox1+/+マウスにおいて実施した。1×109PFUのAd−sFlt−1アデノウイルス又はAd−CMV(対照)アデノウイルスを、尾静脈への注射により、動物に注射した。アデノウイルスを投与した9日後に、マウスを個々に、代謝ケージに24時間入れた。体重、食餌及び水の摂取、並びに尿体積を測定した。尿測定に続き、血液試料を第10日に採取し、動物を安楽死させ、動物の腎臓及び肝臓を収集して、さらなる分析を行った。また、アルギナーゼ阻害剤及び/又はL−アルギニンの補充を使用する、併用処置による回復研究も、妊娠していないHmox1−/−マウス及びHmox1+/+動物において行った。マウスに、ノル−NOHA(Bachem、Bubendorf、スイス)を1日1回腹腔内注射した。Ad−sFlt−1アデノウイルスの注射の1週間前から、研究の終了まで、L−アルギニン(Sigma−Aldrich、St.Louis、MO)を、動物に投与し、この投与は、飲料水に25g/Lを補充することによって行った。
尿中アルブミンを、アルブウエル(Albuwell)−Mキット(Exocell Inc、Philadelphia、PA)を使用して決定した。ヒト及びマウスのsFlt−1、KIM−1及びPlGFのための酵素結合免疫吸着アッセイキットを、R&D Systemsから得、製造元の指定に従って実施した。
試料の調製及びリアルタイム定量的ポリメラーゼ連鎖反応を、以前の記載(Wangら、2013年)に従って実施した。
モノクローナル抗3−ニトロチロシン(1:1000の希釈度、Abcam)、抗アルギニノコハク酸シンターゼ(1:500、Abcam)、抗アルギニノコハク酸リアーゼ(1:500、Abcam)、抗アルギナーゼ−1抗体(1:500、Genetex)、及び抗ベータアクチン(1:10000、Sigma)。
ヒト及びマウスの胎盤組織を、免疫組織化学的検査のために、以前の記載に従って調製した。免疫組織化学的検査を、アルギナーゼ−1、Ass1、Asl及び3−ニトロチロシンに対する抗体を使用することによって実施した。以下の抗体を使用した:(1)抗アルギナーゼ−1抗体(1:50、Genetex);(2)抗アルギニノコハク酸シンターゼ(1:100、Abcam);(3)抗アルギニノコハク酸リアーゼ(1:100、Abcam)、及び(4)抗3−ニトロチロシン(1:100の希釈度、Abcam)。マウス腎臓、又はヒト胎盤の生検から得られた切片を再水和させ、正常血清を使用してブロックし、一次抗体と共に室温で2時間インキュベートした。ベクタステイン(Vectastain)ABCキットを使用することによって、DABを用いて、可視化を実施した。ヘマトキシリンを用いて、スライドを対比染色した。Nikon製の倒立顕微鏡及びイメージプロ−プラス(Image Pro−Plus)画像解析ソフトウエアを使用することによって、染色を分析した。
RNA試料を、上記の記載に従って、マウス腎臓組織から調製し、ARK−Genomics、University of Edinburghに提出し、ARK−Genomicsで、品質の分析が行われた。
腎臓溶解液を精査し、試料を、液体窒素中で急速凍結した。モノクローナル抗3−ニトロチロシン(1:1000の希釈度、Abcam)、抗アルギニノコハク酸シンターゼ(1:500、Abcam)、抗アルギニノコハク酸リアーゼ(1:500、Abcam)、抗アルギナーゼ−1抗体(1:500、Genetex)、及び抗ベータアクチン(1:10000、Sigma)を用いて、ウエスタンブロットを実施した。西洋ワサビペルオキシダーゼとコンジュゲートさせた抗ウサギ又は抗マウスIgG二次抗体(1:1000の希釈度、Transduction Laboratories)を使用した。
結果を、平均±SEMとして示し、複数群間の比較を、分散分析を使用して行った。P<0.05の場合に、差が有意であると報告する。
我々は、重度の妊娠高血圧腎症は、VEGF及びHmox1活性の部分的な喪失に起因して発症するおそれがあるという概念を試験するため、並びにヒトのPE様状態を正確に反映するために、妊娠中の、(単一のHmox1対立遺伝子を担持する)ハプロ不全Hmox1(Hmox1+/−)マウスに、sFlt−1をコードするアデノウイルス(Ad−sFlt−1)の静脈内注射を2回投与することによって、sFlt−1を全身に送達した。我々は、このアプローチを使用して、妊娠中のマウスの血漿中のsFlt−1の顕著な増加を再現した。妊娠高血圧腎症についての診断基準のうちの1つが高血圧であることから、平均動脈血圧(MAP)を、妊娠第18.5日に測定し(図3−1 a)、又は収縮期血圧(SBP)を、埋め込んだ無線遠隔測定装置を連続的に使用して測定した。測定の両方のモードが、E18.5時に、Ad−sFlt−1処置Hmox1+/−群において、それらの野生型同腹仔と比較して、より高い循環型sFlt−1レベルでは、大幅に上昇した血圧を示した。さらに、無線遠隔測定による研究から、SBPの連続的な急速な増加も示され、SBPは、sFlt−1処置Hmox1+/−マウスにおいて、E18.5時に最大に達した。糸球体内皮炎は、重度の妊娠高血圧腎症の主要な特徴であり、広範な母体の内皮損傷の良好な指標である。これらのマウスから得られた腎臓の組織学的分析から、重度の糸球体内皮炎に典型的な分葉及び糸球体の瘢痕化が明らかになった(図3−1 b)。腎臓の損傷と合致して、アルブミンの尿中への排泄もまた、これらのハプロ不全動物において、野生型マウスと比較して有意に増加した(図3−1 c)。腎臓傷害分子−1(重度の妊娠高血圧腎症と関連がある近位尿細菅の傷害に特異的なマーカー)の尿中レベルの増加(図3−1 d)、及び(重度の妊娠高血圧腎症と関連がある)sFlt−1の尿中レベルの上昇(図3−2 e)により、重度の腎臓傷害がさらに立証された。
Claims (12)
- 妊娠中の対象の妊娠高血圧腎症の診断又は予後予測を補助するための方法であって、妊娠中の対象から得られる試料を用意するステップと、試料中の、(a)sFlt−1及びPlGFのうちの一方又は両方の量と、(b)ヘムの分解生成物及びアルギニンの分解生成物のうちの一方又は両方の量との間の比を測定するステップとを含み、測定されるヘムの分解生成物が、ビリルビン、ビリベルジン、一酸化炭素、フェリチン及びカルボキシヘモグロビンから選択される1つ又は複数であり、測定されるアルギニンの分解生成物が、尿素、オルニチン、シトルリン、アルギニノコハク酸又はアンモニアから選択される1つ又は複数である、方法。
- 前記比を所定の比と比較して、妊娠高血圧腎症の診断又は予後予測の決定を補助する、請求項1に記載の方法。
- 試料中でビリルビンの量が測定される、請求項1又は2に記載の方法。
- 試料中で尿素の量が測定される、請求項1〜3のいずれか一項に記載の方法。
- 前記試料が、血液、血清、血漿又は尿である、請求項1〜4のいずれか一項に記載の方法。
- 前記対象における早発性又は遅発性の妊娠高血圧腎症の存在の検出を補助する、請求項1〜5のいずれか一項に記載の方法。
- 試料中のsFlt−1の量、ヘムの分解生成物の量、及び任意選択でPlGFの量、及び任意選択でアルギニンの分解生成物が、イムノアッセイにより決定される、請求項1〜6のいずれか一項に記載の方法。
- (i)sFlt−1及び/若しくはPlGFに特異的な結合剤と、(i)ヘムの分解生成物及び/若しくはアルギニンの分解生成物に特異的な結合剤、並びに/又は(ii)ヘムの分解に特異的なアッセイキットの構成成分を含み、測定されるヘムの分解生成物が、ビリルビン、ビリベルジン、一酸化炭素、フェリチン及びカルボキシヘモグロビンから選択され、測定されるアルギニンの分解生成物が、尿素、オルニチン、シトルリン、アルギニノコハク酸又はアンモニアから選択される、請求項1〜7のいずれか一項に記載の方法における使用のためのアッセイキット。
- 特異的な結合剤が、sFlt−1に又はヘムの分解生成物に特異的な抗体又はその断片を含む、請求項8に記載のアッセイキット。
- ヘムの分解生成物が、ビリルビン、ビリベルジン、カルボキシヘモグロビン又はフェリチンである、請求項8又は9に記載のアッセイキット。
- 妊娠中の対象の妊娠高血圧腎症の診断又は予後予測を補助するための方法を計算するための手段を含むコンピュータであって、前記コンピュータは、(i)妊娠中の対象から得られる試料中のsFlt−1及び/又はPlGFの量を示すシグナル、並びに(ii)妊娠中の対象から得られる試料中のヘムの分解生成物及び/又はアルギニンの分解生成物の量を示すシグナルを受信し、前記シグナルを比較して比を生成するための手段及び前記比を表示するためのディスプレイを有するように構成されたコンピュータであり、測定されるヘムの分解生成物が、ビリルビン、ビリベルジン、一酸化炭素、フェリチン及びカルボキシヘモグロビンから選択され、測定されるアルギニンの分解生成物が、尿素、オルニチン、シトルリン、アルギニノコハク酸又はアンモニアから選択される、コンピュータ。
- 請求項11に記載のコンピュータを備える分析用デバイス。
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