Mathematical Model of Parotid Acinar Differentiation

描述(申请人提供)：拟议项目的长期目标是确定信号网络的发展，该网络可诱导细胞分化为成熟的唾液浆液性腺泡细胞，从而使基因治疗方法能够再生或替换患者的唾液组织。数以百万计的患者由于干燥综合征或放射治疗而丧失唾液腺功能。了解唾液细胞的分化是修复病变或被破坏的腮腺组织的必要步骤。以前的工作已经从组织学上描述了腺泡细胞的末端分化，并通过表征分化标记的表达来描述，但还没有使用基因组水平的方法或数学模型来定义调控途径。目前应用的主要目标是开发正式的数学和统计模型，以识别导致腮腺腺泡细胞终末分化的网络。动态数学模型将用于产生将被检验的假设，并且该模型将通过纳入新数据来反复改进。这项申请是对RFA“唾液腺生物学的系统方法”的响应。我们对这些研究的总体假设是，数学模型可以识别控制腮腺腺泡细胞分化的关键调控途径。特定目标1将使用激光捕获显微切割(LCM)从胚胎和新生大鼠腮腺腺泡细胞中获取RNA，用于微阵列分析分化期间基因表达的模式。我们将建立一个耦合的常微分方程(ODE)模型来描述指导微分过程的假设相互作用。这些假说将通过RT-PCR、IHC和Western blots的组合进行检验，ODE模型也将得到完善。由于microRNAs是发育的重要调节因子，特定的目标2将定义microRNAs在腺泡细胞中的表达，以及分化过程中的变化模式。目前还没有文献描述腮腺组织中的microRNAs。结果将被用来修正微分的数学模型。具体目标3将创建一种统计算法，通过定义偏差和变异的来源以及通过评估模型的总体预测能力及其各个子模块来验证和修订ODE模型。这将允许置信度区间与ODE模型内的不同路径相关联。特定目标4将使用ODE模型对腮腺腺泡细胞中调控基因表达的特定途径做出假设。这些假说将通过转基因和转导实验进行验证，结果将用于完善和验证数学模型。这种系统生物学方法应该确定驱动腮腺腺泡细胞分化的分子途径。项目简介：本研究的总体目标是确定控制细胞分化为分泌性唾液腺泡细胞的分子机制。它满足了数百万因干燥综合征、放射治疗或因基本药物导致的口干症而患有唾液腺功能障碍的美国人的需求。这项研究是开发基因转移治疗和生物制剂等治疗或缓解口腔干燥症症状的新技术的必要基础。

DESCRIPTION (provided by applicant): The long term goal of the proposed project is to define the development of signaling networks that induce differentiation of cells into mature salivary serous acinar cells to allow gene therapy approaches to regenerating or replacing salivary tissue in patients. Millions of patients suffer loss of salivary gland function due to Sj"gren's syndrome or radiation therapy. Understanding the differentiation of salivary cells is a necessary step to enable the restoration of diseased or destroyed parotid salivary tissue. Previous work has described terminal differentiation of acinar cells histologically, and by characterizing the expression of markers of differentiation, but has not used genomics-level approaches, or mathematical models, to define regulatory pathways. The primary goal of the current application is to develop formal mathematical and statistical models that will identify networks which cause terminal differentiation of parotid acinar cells. The dynamic mathematical models will serve to generate hypotheses which will be tested, and the model will be repeatedly refined by the incorporation of new data. This application is in response to the RFA "A Systems Approach to Salivary Gland Biology." Our overall hypothesis for these studies is that a mathematical model can identify key regulatory pathways that control parotid acinar cell differentiation. Specific Aim 1 will use Laser Capture Microdissection (LCM) to obtain RNA from embryonic and newborn rat parotid acinar cells for microarray analysis of the patterns of gene expression across the period of differentiation. A coupled Ordinary Differential Equation (ODE) model will be created to describe the hypothetical interactions that direct the process of differentiation. The hypotheses will be tested, and the ODE model refined, by a combination of RT-PCR, IHC, and Western blots. Since microRNAs are important regulators of development, Specific Aim 2 will define the expression of microRNAs in acinar cells, and the pattern of changes during differentiation. There are currently no publications describing microRNAs in the parotid tissue. The results will be used to revise the mathematical model of differentiation. Specific Aim 3 will create a statistical algorithm to validate and revise the ODE model by defining the sources of bias and variation as well as by assessing the model's predictive power overall, and in its various sub-modules. This will allow confidence intervals to be associated with different pathways within the ODE model. Specific Aim 4 will use the ODE model to make hypotheses about specific pathways regulating gene expression in the parotid acinar cells. These hypotheses will be tested by transfection and transduction experiments, and the results shall be used to refine and validate the mathematical model. This systems biology approach should identify molecular pathways that drive cytodifferentiation of parotid acinar cells. Project Narrative: The overall goal of this research is to define the molecular mechanisms which control differentiation of cells into secretory salivary acinar cells. This addresses the needs of millions of Americans who suffer from salivary gland dysfunction due to Sj"gren's Syndrome, radiation therapy, or xerostomia due to essential medications. This research is a necessary foundation for developing new technologies such as gene transfer therapy and biologics for treating or alleviating the oral symptoms of xerostomia.