Systems biology approach to optimize tissue growth in vitro

描述(申请人提供)：尽管针对体外培养功能性结缔组织进行了大量且不断增加的研究工作，但它们缺乏所需的功能，几乎没有例外，因为组织生长有限，所产生的材料/机械性能不足以在体内应用。虽然单独或联合使用化学和机械信号确实可以改善按材料/机械性能衡量的组织生长，但目前在整个组织生长过程中以固定水平应用这些信号的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，通过组合化学和机械刺激来优化体外组织生长，而不需要了解信号产生和转导机制的知识，这些机制的复杂性令人望而生畏，以至于在可预见的未来无法实现“第一原则”模型。它的灵感来自于Janes和Lauffenburger(Janes，Kelly等人)最近提出的系统生物学方法。基于主成分分析和判别偏最小二乘回归，我们将扩展到确定感兴趣的长期“下游”细胞响应(胶原蛋白和弹性蛋白的产生以及相关的组织机械性能)和由于细胞外刺激而产生的短期“上游”细胞内信号(即磷酸化蛋白质)的子集之间的统计关系。在对细胞进行了一系列长期阶跃响应实验后，确定了这种“信号响应减少”，这些细胞受到不同浓度的化学物质(例如，转化生长因子-β1)和机械(拉伸)刺激，然后将实施“实验性最陡下降”策略，以驱动细胞在生长组织时产生最大数量的胶原和弹性蛋白，这将使组织的强度和模数最大化，同时提供弹性。这将通过在现场周期性地执行短期阶跃响应(询问)实验并使用预定的缩减量来预测后续潜伏期的转化生长因子-β1浓度和拉伸条件的最佳组合来实现，在此之后再次询问组织并且再次更新最佳条件等等。如果成功，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通路。 与公共卫生相关：培育功能性组织替代物的尝试很少成功。为了生长出更坚硬和更强壮的组织，需要一个选择孵化条件的理论基础。在本项目中，将在孵化过程中定期获取高通量的细胞信号数据，以调整孵化条件，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.