Antibody Chip To Study Beta Cell Apoptosis

描述(申请人提供)：胰岛β细胞对葡萄糖稳态是必不可少的，因此它们的数量和分泌功能都受到精确的控制。胰岛β细胞的凋亡是糖尿病疾病的一个标志，预防细胞凋亡是胰岛移植的主要现实问题。关于β细胞中的促有丝分裂和凋亡信号已知很多，并且已知各种信号通路在一个高度复杂的网络中相互作用。一种特定的细胞因子或生长因子经常启动多条信号通路，只有信号的整合才能决定细胞的结果。由于这种复杂性，迫切需要一种能够筛选多个信令级联以确定哪些信令级联在特定的一组条件下运行的方法。我们建议通过抗体阵列和质谱学相结合的方法来创建方法。将选择合适的抗体，并使用SELDI质谱仪作为一种方法，分离它们的标称抗原和任何相关蛋白。我们将准备一系列抗体，通过测试IGF-II途径中的已知成分来报告与β细胞死亡有关的各种信号通路，以验证其有效性。一旦一个有效的阵列可用，它将被用来识别由新型促炎细胞因子PANDER在经历细胞死亡的β细胞中的信号复合体，作为其受体和分子作用未知的凋亡剂的一个例子。这种方法的优点是它不假设关于被测样本的先验知识，它是快速的，可以适应高通量格式，并且不仅允许识别而且允许隔离信令复合体，从而可以发现新的信息。在成功研究了已知和未知的信号通路后，该方法将应用于人类胰岛。来自移植前人类胰岛的信号复合体将被回顾分析，并与移植受者的临床结果相关联，以测试该方法的预测能力。

DESCRIPTION (provided by applicant): Pancreatic beta cells are essential for glucose homeostasis and therefore are subject to precise control of both number and their secretory function. Apoptosis of beta cells is a hallmark of diabetic diseases and preventing apoptosis is a major practical problem in islet transplantation. Much is known about mitogenic and apoptotic signaling in beta cells and the various signaling pathways are known to interact in a highly complex network. A given cytokine or growth factor often initiates multiple signaling pathways, and it is only the integration of signaling which determines the outcome for the cell. Because of this complexity, a method that enables screening of multiple signaling cascades to determine which ones are operating under a particular set of conditions is much needed. We propose to create method through a combination of antibody arrays and mass spectrometry. Suitable antibodies will be selected and used to isolate their nominal antigens plus any associated proteins, using a SELDI mass spectrometer as a method. We will prepare an array of antibodies that report on various signaling pathways involved in beta cell death validate its usefulness by testing known components in the IGF-II pathway. Once a validated array is available it will be used to identify signaling complexes in beta cells undergoing cell death by the novel pro-inflammatory cytokine PANDER, as an example of an apoptotic agent whose receptor and molecular effects are unknown. The advantage to this method is that it does not presume prior knowledge about the sample tested, it is fast, can be adapted to high throughput formats, and allows not only identification but also isolation of signaling complexes, so that new information can be discovered. After both the known and unknown signaling pathways are successfully investigated, the method will be applied to human islets. Signaling complexes from pre-transplant human islets will be analyzed retrospectively and correlated with the clinical outcome in transplant recipients, to test the predictive power of the method.