Systems biology approach to optimize tissue growth in vitro

描述（由申请人提供）：尽管针对体外生长功能性结缔组织进行了大量且不断增加的研究工作，但除少数例外情况外，由于组织生长有限且所得材料/机械性能不足以用于体内应用，因此这些结缔组织缺乏所需的功能性。虽然单独或组合的化学和机械信号确实可以导致如通过材料/机械性质测量的改善的组织生长，但是在组织生长的整个时期以固定水平施加这些信号的当前范例几乎肯定是次优的。拟议项目的愿景是开发一个合理的基础，通过结合化学和机械刺激优化体外组织生长，不需要信号产生和转导机制的知识，这是如此令人生畏的复杂性，“第一原理”模型是不可实现的可预见的未来。它的灵感来自于Janes和Lauffenburger最近提出的系统生物学方法（简，Kelly等，2004年）的基础上，主成分分析和歧视偏最小二乘回归，我们将扩大，以确定长期“下游”细胞反应的利益之间的统计关系（胶原蛋白和弹性蛋白的产生以及相关的组织机械特性）和短期“上游”细胞内信号的子集这些蛋白质是由于细胞外刺激而产生的（即磷酸化蛋白质）。通过对细胞进行的一系列长期阶跃反应实验，确定了这种“信号反应降低”，（例如TGF-b1）和机械（拉伸）刺激，然后将实施“实验最陡下降”策略以驱动细胞生长组织以产生最大量的胶原蛋白和弹性蛋白，其应使组织强度和模量最大化同时赋予弹性。这将通过定期执行短期阶跃响应来实现（“询问”）原位实验，并使用预定的减少来预测随后温育期的TGF-b1浓度和拉伸条件的最佳组合，之后再次询问组织并再次更新最佳条件，等等。这一范例将极大地改变组织工程领域，并可能阐明导致细胞外基质成分沉积的信号传导途径。 公共卫生相关性：很少有尝试种植功能性组织替代品取得成功。为了生长更硬和更强的组织，需要选择孵育条件的基本原理。在本项目中，将在孵育期间定期获得高通量细胞信号数据，以调整孵育条件，从而改善组织生长。

DESCRIPTION (provided by applicant): Despite a large and increasing research effort aimed at growing functional connective tissues in vitro, with few exceptions they lack the required functionality because the tissue growth is limited and the resulting material/mechanical properties are inadequate for in vivo applications. While chemical and mechanical signals, alone or in combination, can indeed lead to improved tissue growth as measured by material/mechanical properties, the current paradigm of applying these signals at fixed levels for the entire period of tissue growth is almost certainly suboptimal. The vision for the proposed project is to develop a rational basis for optimizing in vitro tissue growth via combined chemical and mechanical stimulation that does not require knowledge of the signal generation and transduction mechanisms, which are so daunting in complexity that a "first principles" model is unrealizable for the foreseeable future. It is inspired by a systems biology approach recently advanced by Janes and Lauffenburger (Janes, Kelly et al. 2004) based on principal component analysis and discriminate partial least squares regression that we will extend to determine a statistical relation between long-term "downstream" cell responses of interest (collagen and elastin production and associated tissue mechanical properties) and a subset of short-term "upstream" intracellular signals (i.e. phosphorylated proteins) that are generated due to extracellular stimuli. Having determined this "signal-response reduction" from a series of long-term step-response experiments on cells subject to various concentrations of chemical (e.g. TGF-b1) and mechanical (stretching) stimulation, an "experimental steepest descent" strategy will then be implemented to drive the cells as they grow a tissue to produce the maximum amount of collagen and elastin, which should maximize the tissue strength and modulus while imparting elasticity. This will be accomplished by periodically performing short-term step-response ("interrogation") experiments in situ and using the predetermined reduction to predict the optimal combination of TGF-b1 concentration and stretching conditions for the subsequent incubation period, after which the tissue is again interrogated and the optimal conditions are again updated, and so on. If successful, this paradigm would dramatically change the field of tissue engineering and likely elucidate signaling pathways that lead to deposition of extracellular matrix components. PUBLIC HEALTH RELEVANCE: Few attempts to grow functional tissue replacements have succeeded. In order to grow stiffer and stronger tissues, a rationale is needed to choose the incubation conditions. In this project, high- throughput cell signaling data will be obtained periodically during the incubation to adjust the incubation conditions so that tissue growth is improved.