Quantitative RT/PCR-based radiation dosimetry

如果发生使用放射性材料的恐怖袭击，根据暴露剂量对患者进行风险分层将是确保适当护理的关键。目前的剂量分析需要几天时间才能完成，这会推迟挽救生命的治疗。这项建议寻求利用GeneXpert平台开发基于RNA的剂量学分析，该平台将在辐射暴露后立即对患者进行风险分层。具体目的1.发现体外照射人外周血细胞后出现剂量依赖性表达变化的基因。辐射暴露会引起外周血细胞表达的剂量依赖性变化。我们假设，这些表达变化可以用来开发基于RNA的分析，以区分临床上相关的照射剂量。因此，DNA微阵列将被用来识别体外照射后造血细胞中具有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的表达变化，候选基因将在特定目标2和3中进行检测。特定目标2.确定使用血浆或细胞进行的基于RNA的剂量测定与犬模型中的辐射剂量之间的最佳相关性。受到辐射损伤的细胞死亡，将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的分析在区分辐射剂量方面将更加具体。道德规范 这些问题排除了对人类辐射反应的系统检查，但狗提供了一个最佳的动物模型来检查辐射在体内的影响。因此，将在犬模型中检查来自特定目标1的候选基因的定量RT/PCR分析，以确定哪些基因在体内表现出剂量依赖性的表达变化，以及是否应该使用血浆或血细胞进行剂量测定。然后将使用GeneXpert平台开发剂量分析，这些GeneXpert剂量分析将在更多的狗身上进行验证。具体目的3.确定体外剂量依赖基因是否与移植患者的辐射暴露相关。狗和人类对辐射的反应可能有细微的差异。此外，更长时间的暴露可能会引起不同的辐射反应。我们假设，RNA表达的变化可能有助于确定长时间暴露后的累积照射剂量。因此，定量RT/PCR检测来自中国的候选基因 具体目标1将在移植患者中进行检查，以确定哪些候选基因在接受数天照射的人类中表现出剂量依赖性反应，以及血浆或血细胞是否对人类剂量测定的发展是最佳的。

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.