JP6913977B2 - 前立腺特異膜抗原ベースの前立腺癌患者スクリーニング方法 - Google Patents
前立腺特異膜抗原ベースの前立腺癌患者スクリーニング方法 Download PDFInfo
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Description
前立腺癌患者から血液を取得するステップと、
バイオチップを用いて、前記血液から血中循環癌細胞を分離するステップと、
前記分離された血中循環癌細胞を、血中循環癌細胞に特異的に結合する蛍光マーカーおよび前立腺特異膜抗原に特異的に結合する蛍光マーカーと反応させるステップと、
前記蛍光マーカーと反応させた血中循環癌細胞および前立腺特異膜抗原を対象として、複数の波長範囲のそれぞれに対する光学画像を受信するステップと、
前記複数の波長範囲の全体または一部の光学画像において、前記血中循環癌細胞および前記前立腺特異膜抗原の蛍光強度を測定し、第1フィルタ処理を行うステップと、
前記複数の波長範囲の全体または一部の光学画像において、前記血中循環癌細胞のモルフォロジー(morphology)を測定し、第2フィルタ処理を行うステップと、
前記複数の波長範囲のそれぞれに対する光学画像の全体または一部をマージした統合画像において、前記血中循環癌細胞のモルフォロジーを測定し、第3フィルタ処理を行うステップと、
を含んでいてもよい。
前記複数の波長範囲の全体または一部の光学画像から血中循環癌細胞の大きさを測定するステップと、
前記測定された血中循環癌細胞の大きさよりも所定の割合または量だけ大きい多角形または円形からなる領域を設定し、前記領域内で血中循環癌細胞の蛍光強度を測定し、第1フィルタ処理を行うステップと、を含んでいてもよい。
実験で使用されている高密度マイクロチップは、気孔径(pore−size)が5.5〜8.5μmであるため、5.5μmよりも小さい白血球(WBC:white blood cell)および赤血球(RBC:red blood cell)の場合、チップを通過させて除去し、5.5μmよりも大きい標的細胞の場合、チップを通過させないようにすることで、特定のサイズを選択的に回収できるように考案したマイクロチップである。
1.血液5mlに抗体重合体250μlを入れ、3秒程度混合した後、常温で20分間反応させる。
2.1%FBSを含むPBS5mlを加える。
3.Ficoll溶液15mlが入っている50mlチューブ(tube)に反応溶液10mlを注意深く入れる。
4.溶液を1200gで20分間遠心分離し、血球細胞を1次的に除去する。
5.不要な細胞吸着を防止するために、高密度マイクロチップ(HD Microporous chip、濾過網)を0.1%BSA溶液で10分間処理してコーティングした後、PBSでリンスする。
6.Ficollの上層溶液を濾過網上に置き、微量に存在する赤血球を重力濾過して、高純度の血中循環癌細胞を2次的に分離する。これにより、遠心分離や免疫ビーズ(immunobead)などの処理を行わないことにより、血中循環癌細胞の損傷を防ぐ。
7.分離された血中循環癌細胞は染色により同定される。
本発明による高密度マイクロチップにより分離された血中循環癌細胞を、中性電荷を有する親水性ハイドロゲルでコーティングされた超低付着(Ultra low attachment)培養プレートに播種した。前記培養プレートには、11ng/mlのインスリン、22ng/mlのトランスフェリン、2ng/mlのEGFおよび8μMのROCK阻害剤を含む培養液が含まれており、前記短期培養は、培養した時点から14日間、細胞培養インキュベーターで37℃および5〜10%CO2の条件下で行われた。
前記実施例3により短期培養された血中循環癌細胞が染色法により癌細胞であることを確認するために、下記の方法を用いて細胞染色工程を行う。
2.抗体が細胞内に入ることができるように、透過(permeabilization)工程を行う。
3.PBSで洗浄(washing)工程を行う。
4.PBSを用いて1%BSA(bovine serum albumin:ウシ血清アルブミン)を得、非特異的結合(non−specific binding)および内因性ペルオキシダーゼ活性(endogenous peroxidase activity)を低下させるために、ブロッキング(blocking)工程を行う。
5.1次抗体としてのEpCAM(epithelial cell adhesion molecule)、pan−CK(cytokeratin)、CD45(leukocyte common marker)を、常温で60分間反応させる。
6.前記1次抗体に結合する蛍光標識された二次抗体を、室温で60分間反応させる。
7.PBSで洗浄工程を行う。
8.最後に、細胞核を染色するために、4’,6−ジアミジノ−2−フェニルインドール(DAPI:4’,6−diamidino−2−phenylindole)溶液を入れた後、ガラスカバーを覆い、常温で10分間反応させる。
9.染色された細胞を観察しながら、染色率および回収率をマニュアルで計算する。
1.前記実施例4で蛍光標識された血中循環癌細胞を準備する。
2.前立腺特異膜抗原(Prostate Specific Membrane Antigen(Rabbit mAb,cell signaling technology))に特異的な1次抗体(マウス)を、前記実施例4で蛍光標識された血中循環癌細胞と反応させた後、2次抗体(Goat anti −Rabbit IgG(H+L)Cross−Adsorbed Secondary Antibody、Alexa Fluor 546,Thermo)を反応させて、前立腺特異膜抗原を蛍光標識する。
3.前記反応後の血中循環癌細胞をスライドにロードした後、前記スライドをSmartBiopsy Cell Image Analyzer(CIA 020)のプラットフォーム上に置く。
4.複数の波長範囲を設定して画像を撮影した後、蛍光強度の測定を行う。
5.複数の波長範囲で前記蛍光強度を測定する場合、血中循環癌細胞および前立腺特異膜抗原の蛍光波長を測定して第1フィルタ処理を行う。
6.前記第1フィルタ処理により血中循環癌細胞の細胞核および細胞膜に対して、蛍光マーカー(DAPI、EpCAM、CK、CD45)の蛍光波長(青、緑、赤)を測定する。
7.前記測定された蛍光の波長において、再び血中循環癌細胞を標識した蛍光物質の波長に対して、血中循環癌細胞の面積、細胞の大きさ及び円形度(circularity)を測定する第2フィルタ処理を行う。
8.前記第2フィルタ処理は、前記第1フィルタ処理された画像から血中循環癌細胞の細胞核をさらに測定および分析し、CD45(赤色)で染色された細胞を除く工程を行うことを含む。
9.前記第1フィルタ処理画像及び第2フィルタ処理画像を全体として統合した後、再び血中循環癌細胞の面積、細胞の大きさおよび円形度(circularity)を測定および分析する第3フィルタ処理を行う。
10.各蛍光波長の強度を測定し、これを数値化する。
一般に、細胞培養に使用される細胞培養液および本発明による培養液における血中循環癌細胞の***および成長の度合いを比較実験した。一般に、細胞培養に使用される培養液は、25nM亜セレン酸ナトリウム、50nMヒドロコルチゾン、0.01mMエタノールアミン、0.01mMホスホリルエタノールアミン、100pMトリヨードサイロニン、0.5%(w/v)ウシ血清アルブミン、10mMHEPES、0.5mMピルビン酸ナトリウム、4.5mM L−グルタミンおよび1X抗生物質(antibiotic)−抗真菌剤(antimycotic)を含み、本発明による培養液は、前記実施例3と同様である。また、培養条件は、一般的なプレートを用いたことを除いては、実施例3と同様である。
Claims (10)
- 前立腺癌患者から血液を取得するステップと、
複数の気孔を有し、気孔径(pore−size)が5.5〜8.5μmであるBSA溶液でコーティングされた細胞分離用マイクロチップを用いて、前記血液から血中循環癌細胞を分離するステップと、
前記分離された血中循環癌細胞を、血中循環癌細胞に特異的に結合する蛍光マーカーおよび前立腺特異膜抗原に特異的に結合する蛍光マーカーと反応させるステップと、
前記蛍光マーカーと反応させた血中循環癌細胞および前立腺特異膜抗原を対象として、複数の波長範囲のそれぞれに対する光学画像を受信するステップと、
前記複数の波長範囲の全体または一部の光学画像において、前記血中循環癌細胞および前記前立腺特異膜抗原の蛍光強度を測定し、第1フィルタ処理を行うステップと、
前記複数の波長範囲の全体または一部の光学画像において、前記血中循環癌細胞のモルフォロジー(morphology)を測定し、第2フィルタ処理を行うステップと、
前記複数の波長範囲のそれぞれに対する光学画像の全体または一部をマージした統合画像において、前記血中循環癌細胞のモルフォロジーを測定し、第3フィルタ処理を行うステップと、を含む
ことを特徴とする前立腺癌患者スクリーニング方法。 - 前記血中循環癌細胞に特異的に結合する蛍光マーカーは、ビメンチン(vimentin)に特異的な抗体、上皮細胞接着分子(EpCAM:Epithelial cell adhesion molecule)に特異的な抗体およびサイトケラチン(CK:cytokeratin)に特異的な抗体からなる群から選択される少なくとも1つである
請求項1に記載の前立腺癌患者スクリーニング方法。 - 前記光学画像を受信するステップにおいて、前記複数の波長範囲の光学画像は、青色の波長範囲の画像、緑色の波長範囲の画像、及び赤色の波長範囲の画像を含む
請求項1に記載の前立腺癌患者スクリーニング方法。 - 前記青色の波長範囲の画像において、前記第1フィルタ処理を行うステップおよび前記第2フィルタ処理を行うステップを実行して、血中循環癌細胞の細胞核を判別する
請求項3に記載の前立腺癌患者スクリーニング方法。 - 前記緑色の波長範囲の画像および前記赤色の波長範囲の画像のうちの1つ以上の画像において、前記第1フィルタ処理を行うステップを実行して、血中循環癌細胞の細胞膜を判別する
請求項3に記載の前立腺癌患者スクリーニング方法。 - 前記血中循環癌細胞のモルフォロジーは、細胞の面積、細胞の大きさ、および真円度(circularity)のうちの1つ以上を含む
請求項1に記載の前立腺癌患者スクリーニング方法。 - 前記第1フィルタ処理を行うステップは、
前記複数の波長範囲の全体または一部の光学画像から血中循環癌細胞の大きさを測定するステップと、
前記測定された血中循環癌細胞の大きさよりも所定の割合または量だけ大きい多角形または円形からなる領域を設定し、前記領域内で血中循環癌細胞の蛍光強度を測定し、第1フィルタ処理を行うステップと、を含む
請求項1に記載の前立腺癌患者スクリーニング方法。 - 前記血中循環癌細胞を分離するステップは、大気圧1000〜1020hPa下で行われる
請求項1に記載の前立腺癌患者スクリーニング方法。 - 前記BSA溶液のコーティングは、0.05〜0.15%の濃度で行われる
請求項1に記載の前立腺癌患者スクリーニング方法。 - 前記細胞分離用マイクロチップを用いて分離された血中循環癌細胞を、インスリン、トランスフェリン、EGF、およびROCK阻害剤を含む培地で培養するステップをさらに含む
請求項1に記載の前立腺癌患者スクリーニング方法。
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