生後心筋細胞における細胞周期変化の可視化と核の決意
Summary
To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.
Abstract
Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.
Introduction
心筋細胞の核および細胞周期の状態の正確な同定は、心筋の代謝回転および再生の決意するための極めて重要です。これは、細胞周期活性を同定するための、そのようなpHH3、のKi-67、またはチミジン類似体のような核マーカーの使用のために特に当てはまります。哺乳類成体心筋細胞の増殖能が1非常に小さいので、心筋細胞核の増殖マーカーについて陽性の核の偽の身分証明書は、増殖アッセイの結果で決定的な違いを作ることができます。また、心筋細胞は倍数体および多核細胞ではなく、細胞分裂につながるような核内倍加およびacytokinetic有糸分裂などの細胞周期の変化、になりやすいです。この目的のために、一般的な細胞周期マーカーに対する抗体染色の解釈は、全ての場合において決定的ではありません。
ここで、我々は、直forwaのための方法を提示します RDマウス心筋細胞の認識とそれらの核の明確な同定により生後および成体の段階でネイティブ単離された細胞と厚い組織切片でのnuclearity。そのために、MYH6プロモーターの制御下にヒトヒストンH2BおよびmCherryをからなる融合タンパク質（MYH6-H2B-MCH）の心筋細胞特異的発現を有するトランスジェニックマウス系統は、2を使用しました。 、のeGFP-anillin融合タンパク質の発現は、CMVエンハンサー（CAG-EGFP-anillin）を有するユビキタスニワトリアクチンプロモーターの制御下にあるトランスジェニック増殖インジケータマウス株、このマウス系統を交配することが可能細胞周期状態の決意。足場タンパク質のanillinは、具体的には、アクティブセル3細胞周期で発現され、細胞周期の間に、その差動細胞内局在は、M相の高解像度でライブトラッキング細胞周期の進行を可能にEF "> 4になるので、二重トランスジェニックマウスは、増殖性心筋細胞および細胞周期変動を受けるものとを区別するために使用することができる。これは、in vitroでの増殖を誘導する物質のスクリーニングに特に有用であることを証明。
Protocol
Representative Results
Discussion
心筋細胞は、細胞周期を再入力し、損傷後や組織の恒常性の間に分割することができるかどうかをめぐる論争があります。心筋細胞の基本的な売上高の値は1％1と80％7の範囲で与えられています。 40％7 – 、心臓病変後、細胞周期活性の誘導および新しい心筋細胞の生成は、0.0083％〜8と25の間の値と、境界域で報告されて?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank S. Grünberg (Bonn, Germany) and P. Freitag (Bonn, Germany) for their technical assistance.
Materials
| 10 cm petri dish | Sarstedt | 821472 | |
| 100 µm cell strainer | Becton Dickinson GmbH/Falcon | 352360 | |
| 2,3-Butanedione monoxime (BDM) | Sigma-Aldrich | B0753 | |
| G20x1 ½ injection cannula, Sterican | Braun, Melsungen | 4657519 | |
| 20 gauge needle | Becton Dickinson GmbH | 301300 | |
| 24-well plates | Becton Dickinson GmbH/Falcon | 353047 | |
| 2-Methyl-butane | Carl Roth GmbH + Co. KG | 3927.1 | |
| 37% formaldehyde solution | AppliChem GmbH | A0936,1000 | |
| 3-way stopcock | B. Braun Medical Inc. | 16494C | |
| 50 ml syringe | B. Braun Medical Inc. | 8728810F | |
| 70% ethanol | Otto Fischar GmbH | 27669 | |
| Alexa-Fluor-conjugated secondary antibody | Jackson ImmunoResearch | 115-605-205 | |
| Alpha-Aktinin EA-53, Mouse IgG | Sigma-Aldrich, Steinheim | A7811 | |
| CaCl | Sigma-Aldrich | C4901 | |
| Cell Culture Microplate, 96 Well, Half Area | Greiner bio-one | 675986 | |
| Collagenase B | Roche | 11088815001 | |
| confocal microscope Eclipse Ti-E | Nikon | ||
| cryostat CM 3050S | Leica | ||
| donkey serum | Jackson Immuno Research, Suffolk, GB | 017-000-121 | |
| Dulbecco's Phosphate Buffered Saline | Sigma-Aldrich | D8537 | |
| EDTA | Sigma-Aldrich | E4884 | |
| fetal calf serum | PromoCell, Heidelberg | ||
| Formaldehyde solution (4%) | PanReac AppliChem | A3697 | |
| Gelatine from porcine skin, Type A | Sigma-Aldrich, Steinheim | G2500 | |
| glass coverslips | VWR | 631-0146 | |
| Glucose | Sigma-Aldrich | G7021 | |
| Heidelberger extension tube | IMPROMEDIFORM GmbH | MF 1833 | |
| Heparin-Natrium | Ratiopharm | 5394.02.00 | |
| HEPES | Sigma-Aldrich | H3375 | |
| HistoBond microscope slides | Marienfeld | 0810000 | |
| Hoechst 33342 (1mg/ml) | Sigma Aldrich, Taufkirchen | B2261 | |
| Insulin syringe | Becton Dickinson GmbH | 300334 | |
| Iscove’s ModifiedDulbecco’s Medium (IMDM) | Gibco/Life Technologies, Darmstadt | 21980-032 | |
| KCl | Sigma-Aldrich | P9333 | |
| Laminin | Corning | 354221 | |
| Laser Scanning Mikroskop Eclipse Ti | Nikoninstruments, Düsseldorf | ||
| Lipofectamine RNAiMAX | Invitrogen/Life Technologies, Darmstadt | 13778075 | |
| Mouse IgG Cy5 (donkey) | Jackson ImmunoResearch | 715-175-151 | |
| MGCl | Sigma-Aldrich | M8266 | |
| microcentrifuge tube | Sarstedt | 72690 | |
| Mini shaker | VWR | 12620-940 | |
| mirVana miRNA mimic, hsa-miR199a-3p | Ambion/Thermo Fischer Scientific | 4464066 | |
| Biopsy Mold | Sakura Finetek/ VWR | 4565 | |
| M-slide 8-well ibiTreat | ibidi | 80826 | |
| NaCl | Sigma-Aldrich | S9888 | |
| NaOH | Merck Millipore | 567530 | |
| negative control(scrambled RNA) | Ambion/Thermo Fischer Scientific | AM4611 | |
| Neonatal Heart Dissociation Kit | Miltenyi Biotech, Bergisch Gladbach | 130-098-373 | |
| NIS Elements AR 4.12.01-4.30.02-64bit | Nikoninstruments, Düsseldorf | ||
| Non essential amino acids, NEAA | Gibco/Life Technologies, Darmstad | 11140-035 | |
| Opti-MEM, Reduced Serum Medium | Gibco | 51985-026 | |
| P21-siRNA | Ambion/Thermo Fischer Scientific | 4390771 | |
| P27-siRNA | Ambion/Thermo Fischer Scientific | 4390771 | |
| Penicillin/Streptomycin | Gibco/Life Technologies, Darmstadt | 15140-122 | |
| Phosphate buffered saline (PBS) | Sigma-Aldrich, Steinheim | 14190-094 | |
| Polyvinyl alcohol mounting medium with DABCO®, antifading | Sigma-Aldrich | 10981 | |
| RNase A | Qiagen | 1007885 | |
| RNaseZap | Invitrogen/Life Technologies, Darmstadt | AM9780 | |
| sample containers | Vitlab | 80731 | |
| Serological pipette | Greiner | 607180 | |
| software NIS Elements | Nikon | ||
| Sucrose | Sigma-Aldrich | S0389 | |
| Tissue-Tek O.C.T. Compound | Sakura Finetek/ VWR | 25608-930 | |
| ToPro3 iodide (642/661) | Molecular probes/ThermoFisher Scientific | T3605 | |
| Tris | Sigma-Aldrich | T1503 | |
| Triton X | Fluka | 93418 | |
| Triton X-100 | Fluka | 93418 | |
| Trypsin | Sigma-Aldrich | T1426 | |
| Wheat germ agglutinine (WGA) Fluorescein labeled | Vector Laboratories | VEC-FL-1021-5 | |
| α-actinin antibody | Sigma-Aldrich | A7811 | |
| β-Mercaptoethanol | Sigma-Aldrich, Steinheim | M3148 |
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