新生儿心脏支架：再生研究新矩阵
Summary
在这些研究中，我们提供了新颖，新生儿，鼠心脏的再生研究使用支架的方法。
Abstract
The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.
Introduction
心脏衰竭是常见的，致命的。这是一种渐进性疾病，导致心脏，它损害的血液流向内脏和叶身未满足的代谢需求的收缩力下降。据估计，570万美国人患有心脏衰竭和它是住院在美国9的首要原因。在美国治疗的患者心脏衰竭的集体费用超过$ 300十亿美元，每年9-10。对于终末期心脏衰竭，唯一确定的疗法是心脏移植。每年，估计需要在美国1-2心脏移植程序超过10万个捐助者的心。由于捐助者的数量有限，只有约2,400个移植每年在美国2执行。显然，这个器官短缺需要其他的策略时需出示了transplanta额外的器官加以解决化，理想的是这些器官是自体的，以避免与排斥和寿命免疫抑制有关的并发症。
哺乳动物的成年心肌细胞表现出在损伤有限再生能力，但最近的证据表明哺乳动物新生儿心脏维持损伤后5-8显着的再生能力。具体地讲，下面的部分的手术切除，再生窗口已被生产后天之间发现7.该再生周期的特征是缺乏纤维化瘢痕，形成新血管形成的，从心外膜血管生成因子的释放，和心肌细胞增殖5-8 ，11。这个再生的时间窗口提供了使用新生儿心脏作为材料的生物人工心脏的发展了一种新的源的电位。
细胞外基质是已知的以提供重要的线索以促进cardiomyocytË增殖和生长。在新生儿和成人的矩阵12和促进再生能力的分子的可用性明显的差异已探索13。脱细胞成人基质已在几个研究中用于提供蜂窝复育的ECM支架和生物人工心脏的产生。虽然这些研究，并在干细胞技术的新的发现，正在迅速推进，几个障碍尚未得到满足。例如，在维护基质的天然结构，蜂窝融入矩阵壁的限制，并能够支持增殖和生长都限制了这种方法的成功。而优异的再生属性已被归因于新生儿心脏，使用这样的组织的实际方面限制了它的探索。
根据新生儿心脏的证明再生能力，我们通过开发一个新的发展矩阵脱细胞为P3小鼠心脏的技术。之所以选择这些研究的P3心脏，因为它是心肌再生的窗口内，先前确定的6，但心脏够大丰收，decellularize和recellularize。这项研究的目的是证明创建从新生小鼠心脏的矩阵的可行性。我们的研究为脱细胞一分钟，新生儿心脏同时维持ECM的结构和蛋白质的完整性的可行性提供了证据。我们也展示给recellularize与mCherry表达心肌这种心脏ECM的能力，我们已经研究这些心肌细胞为以下recellularization各种心脏标志物的表达。该技术将允许用于生物人工心脏的发展新生儿矩阵的优越性的测试。
Protocol
Representative Results
Discussion
这种技术对心脏的重复灌注的依赖使得栓塞的回避取得成果的一个重要组成部分。从心脏在步骤2.2-2.6初始导尿，到步骤2.8-2.14之间溶液的变化，有操作，可以允许引入气泡而妥协灌注液流入心肌的。由于新生儿心脏的面积小，在血管甚至微小气泡可引起心肌梗死的技术，从而使脱细胞不完整的。另外，在以后的洗涤步骤中，不完整的灌注可导致洗涤剂残留有负面影响的生物相容性。此外，如在步?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors gratefully acknowledge Ms. Cynthia DeKay for the preparation of the figures.
Materials
| 1. Materials For Mouse Heart Isolation | |||
| P1 mouse pups (as shown; B6;D2-Tg(Myh6*-mCherry)2Mik/J) | Jackson Laboratories | 21577 | or equivalent |
| 60 mm Culture dish | BD Falcon | 353004 | or equivalent |
| Phosphate buffered saline pH 7.4 (sterile) | Hyclone | SH30256.01 | or equivalent |
| Single Use Blade | Stanley | 28-510 | or equivalent |
| Standard Scissors | Moria Bonn (Fine Science Tools) | 14381-43 | or equivalent |
| Spring Scissors 10 cm | Fine Science Tools | 15024-10 | or equivalent |
| Vannas Spring Scissors – 3mm Cutting Edge | Fine Science Tools | 15000-00 | or equivalent |
| #5 Forceps | Dumnot (Fine Science Tools) | 11295-00 | or equivalent |
| 2. Materials For Decellularization | |||
| Inlet adaptor | Chemglass | CG-1013 | autoclavable |
| Septum | Chemglass | CG-3022-99 | autoclavable |
| 1/8 in. ID x 3/8 OD C-Flex tubing | Cole-Parmer | EW-06422-10 | autoclavable |
| Male luer to 1/8" hose barb adaptor | McMaster-Carr | 51525K33 | autoclavable |
| Female luer to 1/8" hose barb adaptor | McMaster-Carr | 51525K26 | autoclavable |
| Prolene 7-0 surgical suture | Ethicon | 8648G | or equivalent |
| Ring stand | Fisher Scientific | S47807 | or equivalent |
| Clamp | Fisher Scientific | 05-769-6Q | or equivalent |
| Clamp regular holder | Fisher Scientific | 05-754Q | or equivalent |
| 60 cc syringe barrel | Coviden | 1186000777T | or equivalent |
| Beaker | Kimble | 14000250 | or equivalent |
| 22g x 1 Syringe Needle | BD | 305155 | or equivalent |
| 12 cc syringe | Coviden | 8881512878 | or equivalent |
| 3-way stop cock | Smith Medical | MX5311L | or equivalent |
| 22 x 1 g needle | BD | 305155 | or equivalent |
| PE50 tubing | BD Clay Adams Intramedic | 427411 | Must be formable by heat. Polyethylene recommended |
| 1% SDS | Invitrogen | 15525-017 | Ultrapure grade recommended. Make up fresh solution and filter sterilize before use. |
| 1% Triton X-100 | Sigma-Aldrich | T8787 | Make up fresh solution from a 10% stock and filter sterilize before use. |
| Sterile dH2O | Hyclone | SH30538.02 | Or MilliQ system purified water. |
| 1X Pen/Strep | Corning CellGro | 30-001-Cl | or equivalent |
| 3. Materials For DNA Quantitation | |||
| Proteinase K | Fisher | BP1700 | >30U/mg activity |
| KCl | Sigma-Aldrich | P9333 | or equivalent |
| MgCl*6H2O | Mallinckrodt | 5958-04 | or equivalent |
| Tween 20 | Sigma-Aldrich | P1379 | or equivalent |
| Tris base/hydrochloride | Sigma-Aldrich | T1503/T5941 | or equivalent |
| Pico-Green dsDNA assay kit | Life Technologies | P7589 | requires fluorimeter to read |
| 4. Method for fixation and sectioning of tissue. | |||
| Paraformaldehyde | Sigma-Aldrich | P6148 | or equivalent |
| Gelatin Type A from porcine skin | Sigma-Aldrich | G2500 | must be 300 bloom or greater |
| 5. Method for tissue histology | |||
| Cryomolds 10 x 10 x 5mm | Tissue-Tek | 4565 | or equivalent |
| Cryostat | Hacker/Bright | Model OTF | or equivalent |
| Microscope Slides 25 x 75 x 1 mm | Fisher Scientific | 12-550-19 | or equivalent |
| Hematoxylin 560 | Surgipath/Leica Selectech | 3801570 | or equivalent |
| Ethanol | Decon Laboratories | 2701 | or equivalent |
| Define | Surgipath/Leica Selectech | 3803590 | or equivalent |
| Blue buffer | Surgipath/Leica Selectech | 3802915 | or equivalent |
| Alcoholic Eosin Y 515 | Surgipath/Leica Selectech | 3801615 | or equivalent |
| Formula 83 Xylene substitute | CBG Biotech | CH0104B | or equivalent |
| Permount Mounting Medium | Fisher Chemical | SP15-500 | or equivalent |
| Collagen IV Antibody | Rockland | 600-401-106.1 | or equivalent |
| α-Actinin Antibody | Abcam | AB9465 | or equivalent |
| mCherry Antibody | Abcam | AB205402 | or equivalent |
| NKX2.5 Antibody | Santa Cruz Biotechnology | SC-8697 | or equivalent |
| Donkey anti-mouse AF488 Antibody | Life Technology | A21202 | or equivalent |
| Donkey anti-chicken AF594 Antibody | Jackson Immunoresearch | 703-585-155 | or equivalent |
| Donkey anti-goat CY5 Antibody | Jackson Immunoresearch | 705-175-147 | or equivalent |
| Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594 | Jackson Immunoresearch | 111-587-003 | or equivalent |
| Prolong Gold Antifade Mountant with DAPI | ThermoFisher | P36930 | or equivalent |
| 6. Isolation of neonatal ventricular cardiomyocytes using pre-plating. | |||
| HBSS (Ca, Mg Free) | Hyclone | SH30031.02 | or equivalent |
| HEPES (1M) | Corning CellGro | 25-060-Cl | or equivalent |
| Cell Strainer | BD Falcon | 352340 | or equivalent |
| 50 mL tube | BD Falcon | 352070 | or equivalent |
| Primeria 100 mm plates | Corning | 353803 | Primeria surface enhances fibroblast attachment promoting a higher myocyte purity |
| Trypsin | Difco | 215240 | or equivalent |
| DNase II | Sigma-Aldrich | D8764 | or equivalent |
| DMEM (Delbecco's Minimal Essential Media) | Hyclone | SH30022.01 | or equivalent |
| Vitamin B12 | Sigma-Aldrich | V6629 | or equivalent |
| Fibronectin coated plates | BD Bioscience | 354501 | or equivalent |
| Fetal bovine serum | Hyclone | SH30910.03 | or equivalent |
| Heart bioreactor glassware | Radnoti Glass Technology | 120101BEZ | Must be sterilizable by autoclaving or gas. |
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