细胞周期变化的可视化和核测定心肌出生后
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
心肌细胞核和细胞周期状态的正确识别是对心肌的营业额和再生的决心至关重要。这是一种使用核标记物，如pHH3，Ki-67的，或胸苷类似物，用于识别细胞周期活动的情况尤其如此。如成年哺乳动物的心肌细胞的增殖能力是非常小的图1中 ，核阳性心肌细胞核的增殖标志物可以使在一个增殖试验的结果的一个关键差的误识别。此外，心肌细胞很容易在细胞周期的变化，如核内复制和acytokinetic有丝分裂，这导致多倍体和多核细胞，而不是在细胞分裂。为此，对常见的细胞周期标记物的抗体染色的解释不是在所有情况下定论。
在这里，我们提出了直forwa方法小鼠心肌细胞的RD承认和其核的明确的身份标识在产后和成年阶段的本土分离出的细胞和厚厚的组织切片的nuclearity。为此目的，与MYH6启动子（MYH6-H2B-MCH）的控制下，由人类组蛋白H2B和mCherry融合蛋白的特异性心肌表达的转基因小鼠品系使用2。杂交育种用的转基因增殖指示剂鼠标线这个鼠标线，其中一个的eGFP-anillin融合蛋白的表达是与CMV增强无处不鸡肌动蛋白启动子的控制下（CAG-EGFP-anillin），允许测定的细胞周期状态。支架蛋白anillin在细胞周期特异性表达的活性细胞3，和在细胞周期中其差的亚细胞定位允许活跟踪细胞周期进程用高分辨率的M-相EF“> 4，因此，双转基因小鼠可用于增殖心肌细胞和那些经历细胞周期的变化来区分。这证明在筛选用于体外增殖诱导物质特别有用。
Protocol
Representative Results
Discussion
有一个争论的心肌细胞是否能够重新进入细胞周期和损伤后和组织稳态期间分割。对于心肌细胞的基本成交值已被赋予在范围为1％1和80％的7之间。还心脏损伤后，细胞周期活性的诱导和新的心肌细胞的产生已经报道在边界区，具有值0.0083％8和25之间- 40％7。这些差异可部分通过不同的实验方法进行说明，以确定心肌细胞核，…
Divulgations
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 |
References
- Bergmann, O., et al. Evidence for cardiomyocyte renewal in humans. Science. 324 (5923), 98-102 (2009).
- Raulf, A., et al. Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status. Basic Res.Cardiol. 110 (3), 33 (2015).
- Field, C. M., Alberts, B. M. Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. J.Cell Biol. 131 (1), 165-178 (1995).
- Hesse, M., et al. Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle. Nat.Commun. 3, 1076 (2012).
- Eulalio, A., et al. Functional screening identifies miRNAs inducing cardiac regeneration. Nature. 492 (7429), 376-381 (2012).
- Di, S. V., Giacca, M., Capogrossi, M. C., Crescenzi, M., Martelli, F. Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle. J.Biol.Chem. 286 (10), 8644-8654 (2011).
- Hosoda, T., et al. Clonality of mouse and human cardiomyogenesis in vivo. Proc.Natl.Acad.Sci.U.S.A. 106 (40), 17169-17174 (2009).
- Soonpaa, M. H., Field, L. J. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am.J.Physiol. 272 (1 Pt 2), H220-H226 (1997).
- Ang, K. L., et al. Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart. Am.J.Physiol Cell Physiol. 298 (6), C1603-C1609 (2010).
- Engel, F. B., Schebesta, M., Keating, M. T. Anillin localization defect in cardiomyocyte binucleation. J Mol Cell Cardiol. 41 (4), 601-612 (2006).
- Bergmann, O., et al. Dynamics of Cell Generation and Turnover in the Human Heart. Cell. 161 (7), 1566-1575 (2015).
