Mathematical Model of Parotid Acinar Differentiation

描述（由申请人提供）：拟议项目的长期目标是确定诱导细胞分化为成熟唾液浆液性腺泡细胞的信号网络的开发，以允许基因治疗方法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。了解唾液腺细胞的分化是使病变或破坏的腮腺唾液组织恢复的必要步骤。以前的工作已经描述了终端分化的腺泡细胞的组织学，并通过表征分化标志物的表达，但没有使用基因组学水平的方法，或数学模型，来定义调节途径。本申请的主要目标是开发正式的数学和统计模型，其将识别引起腮腺腺泡细胞的终末分化的网络。动态数学模型将用于产生将被检验的假设，模型将通过纳入新数据而反复完善。这个应用程序是响应RFA的“唾液腺生物学的系统方法”。“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。具体目标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得RNA，用于跨分化期基因表达模式的微阵列分析。一个耦合的常微分方程（ODE）模型将被创建来描述指导微分过程的假设相互作用。将通过RT-PCR、IHC和Western印迹的组合来检验假设，并完善ODE模型。由于microRNA是发育的重要调节因子，因此特异性目标2将定义腺泡细胞中microRNA的表达以及分化期间的变化模式。目前没有出版物描述腮腺组织中的microRNA。研究结果将用于修正微分数学模型。具体目标3将创建一个统计算法，通过定义偏差和变异的来源以及评估模型的整体预测能力及其各个子模块来验证和修订ODE模型。这将允许置信区间与ODE模型中的不同途径相关联。具体目标4将使用ODE模型来假设腮腺腺泡细胞中调控基因表达的特定途径。这些假设将通过转染和转导实验进行测试，结果将用于完善和验证数学模型。这种系统生物学的方法应该确定分子途径，推动腮腺腺泡细胞的细胞分化。项目叙述：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。这解决了数百万美国人的需要，他们因干燥综合征、放射治疗或因基本药物引起的口腔干燥症而患有唾液腺功能障碍。该研究为开发基因转移治疗和生物制剂等新技术治疗或缓解口腔干燥症的口腔症状奠定了必要的基础。

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.