Antibody Chip To Study Beta Cell Apoptosis

描述（由申请方提供）：胰腺β细胞对于葡萄糖稳态是必需的，因此其数量及其分泌功能均受到精确控制。β细胞凋亡是糖尿病的一个重要标志，防止胰岛细胞凋亡是胰岛移植中的一个重要实际问题。关于β细胞中的促有丝分裂和凋亡信号传导已知很多，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启动多个信号传导途径，并且仅信号传导的整合决定细胞的结果。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特定条件下运行的方法。我们建议通过抗体阵列和质谱的组合来创建方法。将选择合适的抗体，并使用SELDI质谱仪作为方法，用于分离其标称抗原加上任何相关蛋白质。我们将制备一系列抗体，报告β细胞死亡中涉及的各种信号通路，通过测试IGF-II通路中的已知组分来验证其有效性。一旦经过验证的阵列可用，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，它不假定关于所测试的样品的先验知识，它是快速的，可以适应于高通量格式，并且不仅允许识别而且还允许分离信号传导复合物，使得可以发现新的信息。在成功研究已知和未知的信号通路之后，该方法将应用于人类胰岛。将回顾性分析来自移植前人胰岛的信号复合物，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.