Antibody Chip To Study Beta Cell Apoptosis

描述（由申请人提供）：胰腺β细胞对于葡萄糖稳态至关重要，因此需要对其数量及其分泌功能进行精确控制。 β细胞凋亡是糖尿病疾病的标志，防止细胞凋亡是胰岛移植中的一个主要实际问题。人们对β细胞中的有丝分裂和凋亡信号传导了解很多，并且已知各种信号传导途径在高度复杂的网络中相互作用。给定的细胞因子或生长因子通常会启动多个信号传导途径，并且只有信号传导的整合才能决定细胞的结果。由于这种复杂性，非常需要一种能够筛选多个信号级联以确定哪些信号级联在一组特定条件下运行的方法。我们建议通过结合抗体阵列和质谱来创建方法。将选择合适的抗体并使用 SELDI 质谱仪作为方法来分离其标称抗原和任何相关蛋白。我们将制备一系列抗体，报告参与 β 细胞死亡的各种信号传导途径，并通过测试 IGF-II 途径中的已知成分来验证其有用性。一旦获得经过验证的阵列，它将用于识别通过新型促炎细胞因子 PANDER 进行细胞死亡的 β 细胞中的信号传导复合物，作为其受体和分子效应未知的凋亡剂的一个例子。该方法的优点在于，它不假定有关测试样本的先验知识，速度快，可以适应高通量格式，并且不仅允许识别而且还可以隔离信号复合物，从而可以发现新信息。在成功研究已知和未知信号通路后，该方法将应用于人类胰岛。将回顾性分析来自移植前人类胰岛的信号复合物，并将其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.