Quantitative RT/PCR-based radiation dosimetry

在使用放射性物质的恐怖袭击事件中，根据暴露剂量对患者进行风险分层对于确保适当护理至关重要。目前的剂量测定需要数天才能完成，这将延误挽救生命的治疗。该提案旨在利用GeneXpert平台开发基于rna的剂量测定方法，以便在辐射暴露后立即对患者进行风险分层。具体目标发现在人外周血细胞体外辐照后表现出剂量依赖性表达变化的基因。辐射暴露引起外周血细胞剂量依赖性表达变化。我们假设这些表达变化可以用来开发基于rna的检测方法，以区分临床相关的辐照剂量。因此，DNA微阵列将用于鉴定体外辐照后造血细胞中剂量依赖性表达变化的基因。定量RT/PCR分析将验证这些剂量依赖性表达变化，候选基因将在Specific Aims 2和3中进行检查。具体目标2。确定在犬模型中，使用血浆或细胞进行的rna剂量测定是否与辐射剂量最相关。受辐射损伤的细胞死亡，RNA泄漏到细胞外空间，这种RNA可以通过定量RT/PCR检测到。我们假设使用血浆的基于rna的测定将在区分辐射剂量方面更加具体。道德

In the event of a terrorist attack using radioactive material, risk-stratification of patients based on exposure doses will be critical to ensure appropriate care. Current dosimetry assays take days to perform, which would delay life-saving therapies. This proposal seeks to develop RNA-based dosimetry assays using the GeneXpert platform that will risk-stratify patients immediately after radiation exposures. Specific Aim 1. Discover genes that display dose-dependent expression changes after in vitro irradiation of human peripheral blood cells. Radiation exposures cause dose-dependent expression changes in peripheral blood cells. We hypothesize that these expression changes can be utilized to develop RNA-based assays that discriminate between clinically relevant doses of irradiation. Therefore, DNA microarrays will be used to identify genes with dose-dependent expression changes in hematopoietic cells after in vitro irradiation. Quantitative RT/PCR assays will validate these dose-dependent expression changes, and candidate genes will be examined in Specific Aims 2 and 3. Specific Aim 2. Determine whether RNA-based dosimetry assays using plasma or cells best correlate with radiation doses in the canine model. Cells injured from radiation die, leaking RNA into the extracellular space, and this RNA can be detected using quantitative RT/PCR assays. We hypothesize that RNA-based assays using plasma will be more specific in discriminating between radiation doses. Ethical issues preclude systematic examinations of radiation responses in humans, but dogs offer an optimal animal model to examine the in vivo effects of irradiation. Therefore, quantitative RT/PCR assays for candidate genes from Specific Aim 1 will be examined in the canine model to determine which genes display in vivo dose-dependent expression changes and whether plasma or blood cells should be used for the development of dosimetry assays. Dosimetry assays will then be developed using the GeneXpert platform, and these GeneXpert dosimetry assays will be validated in additional dogs. Specific Aim 3. Determine if in vitro dose-dependent genes correlate with radiation exposure in transplant patients. There may be subtle differences in radiation-induced responses between dogs and humans. In addition, exposure over more extended periods of time may invoke different radiation responses. We hypothesize that RNA expression changes may be useful in determining the cumulative dose of irradiation after prolonged exposures. Therefore, quantitative RT/PCR assays for candidate genes from Specific Aim 1 will be examined in transplant patients in order to determine which candidate genes display dose-dependent responses in humans receiving irradiation over several days and whether plasma or blood cells are optimal for the development of dosimetry assays in humans.