Magnitude Improvement of Molecular Signaling Imaging (RMI)

描述(由申请人提供)：最近对细胞信号通路的定义为多细胞生物协调转录对环境变化的反应提供了新的见解。对哺乳动物的细胞信号进行成像将提供对体内调节这些信号的因素的洞察。电子顺磁共振(EPR)成像在小鼠体内提供了新颖的、高分辨率的氧气图像。光学探测的分子成像具有有限的深度敏感性，这使得在较大动物中进行测量变得困难。其他技术受到各种限制。我们建议探索低频EPR成像，在组织深处具有灵敏度，作为细胞信号的一个例子，检测缺氧肽信号分子的诱导。EPR图像的独特功能包括低信号背景、小型哺乳动物的高空间分辨率(1 mm或更低)、有望扩展到较大的动物以及对动物组织深层的敏感性，这些特点使这项技术与众不同，并鼓励其探索。低氧诱导因子(HIF)蛋白在控制和协调细胞和生物对低氧的反应中发挥着重要作用。这项工作将包括构建EPR自旋探针，注入动物体内，动物在遇到蛋白酶(MMP2)之前保持沉默。它还将涉及将HIF1α结合蛋白启动子上游的MMP2基因构建成病毒载体。然后，病毒载体将被用来1)感染肿瘤细胞或2)正常组织周围的细胞。肿瘤或组织缺氧会刺激HIF-1α结合蛋白的产生，而HIF-1α结合蛋白会刺激MMP2的产生，从而激活沉默的自旋探针。然后将获得EPR氧气图像，以关联在活动物的肿瘤中触发低氧细胞信号的低氧水平。这将提供体内缺氧信号阈值的测量。这种技术有望使分辨率和灵敏度提高数量级。

DESCRIPTION (provided by applicant): The recent definition of cellular signaling pathways has provided new insight into the means by which multi-cellular organisms coordinate a transcriptional response to environmental change. Imaging cellular signaling in mammals will provide insight into the factors that modulate these signals in vivo. Electron Paramagnetic Resonance (EPR) imaging has provided novel, high resolution oxygen images in mice. Molecular imaging with optical detection has limited depth sensitivity making measurements in larger animals difficult. Other techniques suffer from various limitations. We propose here to explore low frequency EPR imaging, with sensitivity deep in tissues, to detect, as an example of cellular signaling, the induction of hypoxia peptide signal molecules. Unique features of EPR images including low signal background, high spatial resolution (1 mm or less) in small mammals, the promise of scaling to larger animals and sensitivity deep in animal tissues distinguish this technique and encourage its exploration. Hypoxia Inducible Factor (HIF) proteins play a major role in controlling and coordinating the response of cells and organisms to hypoxia. The work will involve the construction of an EPR spin probe, to be injected into the animal, which is silent until it encounters a proteinase (MMP2). It will also involve the engineering of an MMP2 gene construct just upstream of the HIF1alpha binding protein promoter into a viral vector. The viral vector will then be used to 1) infect cells in tumors or 2) surrounding normal tissues. Tumor or tissue hypoxia will stimulate production of HIF-1alpha binding protein which will stimulate the production of MMP2 which will activate the silent spin probe. EPR oxygen images will then be obtained to correlate the levels of hypoxia at which hypoxic cell signaling is triggered in the tumors of a living animal. This will provide a measure of the in vivo threshold for hypoxic signaling. Such a technology promises order of magnitude improvements of resolution and sensitivity