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Development of a Long-term, Working Cardiovascular Tissue Slice Culture

长期有效的心血管组织切片培养的发展

基本信息

批准号：
8067879
负责人：
Joseph S Janicki
金额：
$ 17.13万
依托单位：
UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8067879
关键词：
Adult Aging Animals Apoptosis Atrophic Biochemical Biological Biological Assay Cardiac Cardiac Myocytes Cardiovascular system Cell Culture Techniques Cell Size Cells Chronic Coculture Techniques Complex Confounding Factors (Epidemiology)Contracts Creatine Kinase Culture Media Cultured Cells Data Collection Development Disease Ensure Environment Evaluation Excision Extracellular Matrix Female Functional disorder Gases Gene Expression Heart Heart Contractilities Heart Rate Heart failure Histology Hour Human Hypertension Individual Influentials Injury Intervention Isometric Contraction Kidney Lactate Dehydrogenase Lead Left Liver Long-Term Effects Maintenance Mechanics Mitral Valve Insufficiency Molecular Mus Muscle Muscle Cells Myocardial Myocardial tissue Myocardium Neurosciences Research Organ Organ Culture Techniques Papillary Performance Phenotype Physiological Preparation Prevention Process Rattus Research Slice Staging Stimulus Stress Stretching System Testing Time Tissue Viability Tissues Variant Ventricular Work adenylate aged brain tissue cell preparation cell type design extracellular high reward high risk in vivo innovation male novel prevent prototype public health relevance quantum response tissue culture tripolyphosphate

项目摘要

DESCRIPTION (provided by applicant): Our overall objective is to develop a long-term, culture system for adult rat left ventricular tissue slices whereby myocyte atrophy is prevented by continually pacing the slices to contract isometrically. This approach will bridge the gap between the adult isolated heart preparation and cell culture allowing for a greater degree of control, long term stability and viability and a relatively undisturbed extracellular environment. We are working on the principle that if the adenylate triphosphate pool (ATP) is preserved throughout the sequential stages of organ excision, tissue slicing, and ventricular slice incubation, we can preserve the viability of the tissue slices for up to 7 days, if not longer. The advantages to such a system include: 1) conditions in which all myocardial cells retain their native three-dimensional structural integrity and intercellular interactions; 2) ability to perform multiple perturbations on an individual heart, thereby minimizing biological variability; and 3) relatively long term effects of a perturbation can be investigated under highly controlled conditions. Also, the myocardial response would be solely intrinsic in that, regardless of the intervention, it would be void of reflexive adjustments in ventricular loading conditions, heart rate and contractility and variations in neurohormonal background. Most studies using myocardial tissue slices have been limited to several hours of data collection with no attempt to ensure thin, uniform slices, to optimize culture conditions and to stimulate contractions. In order to extend the viability of the tissue slice, our strategy is to establish optimal conditions for viability in a stepwise fashion. To prevent myocyte atrophy, we will test two prototype systems which are designed to impose a physiologic myocardial stretch (preload) and to stimulate the slice to contract isometrically. We will evaluate tissue slice functional viability along with assays for gene expression, apoptosis, ischemic damage (histology or biochemical assays for ATP and lactate dehydrogenase), myocyte atrophy (myocyte cell size), and myocyte injury (release of creatine kinase-MB into tissue slice culture media). There are unlimited applications associated with the proposed system that would be relevant to each cardiac cell type and their interactions with each other and the extracellular environment. Clearly, such a preparation has tremendous potential to contribute significantly to our understanding of mechanisms at the molecular, cellular and tissue levels in normal, genetically modified, aged, male and female, and diseased hearts. Thus, while this is a high- risk undertaking, it would become a high-reward endeavor if successful. PUBLIC HEALTH RELEVANCE: The cardiac remodeling that occurs in response to hypertension, mitral valve regurgitation or heart muscle injury will eventually lead to heart failure. There is a critical need for a cardiac tissue slice culture system that remains alive for up to 7 days in order to investigate at the molecular, cellular and tissue levels the mechanisms that cause the heart to fail. To develop such a system is the objective of this project.

描述（由申请人提供）：我们的总体目标是开发一种用于成年大鼠左心室组织切片的长期培养系统，通过持续起搏切片以等距收缩来防止肌细胞萎缩。这种方法将弥合成人分离心脏制备物和细胞培养物之间的差距，从而允许更大程度的控制、长期稳定性和活力以及相对不受干扰的细胞外环境。我们的工作原理是，如果在器官切除、组织切片和心室切片孵育的整个顺序阶段中保留三磷酸腺苷池（ATP），我们可以将组织切片的活力保存长达7天，如果不是更长的话。这种系统的优点包括：1）所有心肌细胞保持其天然三维结构完整性和细胞间相互作用的条件; 2）对单个心脏进行多次扰动的能力，从而使生物变异性最小化;以及3）可以在高度受控的条件下研究扰动的相对长期影响。此外，心肌反应将是完全内在的，因为无论干预如何，它将不会对心室负荷条件、心率和收缩力以及神经激素背景的变化进行反射性调整。大多数使用心肌组织切片的研究仅限于几个小时的数据收集，没有尝试确保薄而均匀的切片，优化培养条件和刺激收缩。为了延长组织切片的生存能力，我们的策略是以逐步的方式建立最佳生存条件。为了防止肌细胞萎缩，我们将测试两个原型系统，这两个原型系统被设计为施加生理性心肌拉伸（预负荷）并刺激切片等距收缩。我们将沿着基因表达、细胞凋亡、缺血性损伤（ATP和乳酸脱氢酶的组织学或生物化学测定）、肌细胞萎缩（肌细胞大小）和肌细胞损伤（肌酸激酶-MB释放到组织切片培养基中）的测定来评价组织切片功能活力。存在与所提出的系统相关联的无限应用，其将与每种心脏细胞类型以及它们与彼此和细胞外环境的相互作用相关。显然，这种制剂具有巨大的潜力，有助于我们理解正常、转基因、老年、男性和女性以及患病心脏的分子、细胞和组织水平的机制。因此，虽然这是一项高风险的事业，但如果成功，它将成为一项高回报的奋进。公共卫生关系：高血压、二尖瓣返流或心肌损伤引起的心脏重构最终会导致心力衰竭。为了在分子、细胞和组织水平上研究导致心脏衰竭的机制，迫切需要保持存活长达7天的心脏组织切片培养系统。开发这样一个系统是本项目的目标。