Systems biology approach to optimize tissue growth in vitro

描述（由申请人提供）：尽管在体外培养功能性结缔组织方面进行了大量且不断增加的研究工作，但除了少数例外，它们缺乏所需的功能，因为组织生长有限，所得材料/机械性能不足以用于体内应用。虽然化学和机械信号，单独或联合使用，确实可以通过测量材料/机械性能来改善组织生长，但目前在组织生长的整个时期以固定水平应用这些信号的范例几乎肯定是不理想的。该项目的愿景是为通过化学和机械联合刺激优化体外组织生长开发一个合理的基础，而不需要了解信号产生和转导机制，这些机制非常复杂，“第一原理”模型在可预见的未来是无法实现的。它的灵感来自于最近由james和Lauffenburger提出的系统生物学方法(james，Kelly et al. 2004)基于主成分分析和判别偏最小二乘回归，我们将对其进行扩展，以确定长期“下游”细胞反应（胶原蛋白和弹性蛋白的产生以及相关的组织力学性能）与细胞外刺激产生的短期“上游”细胞内信号（即磷酸化蛋白）子集之间的统计关系。通过对细胞进行一系列长期的阶梯反应实验，在不同浓度的化学（如TGF-b1）和机械（拉伸）刺激下，确定了这种“信号反应减少”，然后将实施“实验最陡下降”策略，以驱动细胞在生长组织时产生最大数量的胶原蛋白和弹性蛋白，从而在赋予弹性的同时最大限度地提高组织强度和模量。这将通过定期在原位进行短期的步进反应（“询问”）实验来完成，并使用预定的还原来预测随后孵育期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，以此类推。如果成功，这种模式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通路。

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.