Measuring Cytogenetic Damage in Human Blood

人类通过各种来源接触到基因毒剂。微核(MN)的形成是一个终点，可以用来检测由碎裂或非正常发育机制引起的DNA损伤。一个灵敏和高通量的系统来测量人体血液中的MN将有无数的生物监测应用。例如，这样的系统可以提供关于正在进行临床试验的新药、化疗方案以及意外辐射或化学暴露的染色体破坏活性的信息。目前应用的一个目标是优化在第一阶段开发的方法，这些方法允许流式细胞术测量人网织红细胞(RETs0。计划进行实验，以测试MN-RET测量是否代表细胞遗传学损伤的敏感指标。为了实现这一目标，我们的第二阶段目标是。1.完成细胞染色和处理程序的优化。2.制定生物学标准，以辅助流式细胞仪参数的校准。3.测定放疗和放化疗患者治疗前后MN-RET诱导的时程和程度。4.测定放疗和放化疗患者治疗前后MN-RET诱导的时程和程度。5.与研究人员合作，对切尔诺贝利清算人、清理工人和定居者进行环境生物监测研究。拟议的商业应用：该研究项目的成功完成将使Litron实验室成为一家专家设施，能够以付费服务的基础对人类血液样本进行细胞遗传学测量。此外，通过将必要的试剂开发成试剂盒形式，将有可能将这项技术提供给其他能够使用单一激光流式细胞仪的实验室。这项技术创新技术有可能成为一种重要的临床工具，用于测量环境暴露带来的DNA损伤，环境暴露包括放射和/或化疗、衰老和无数其他可能的来源。

Humans are exposed to genotoxic agents through a variety of sources. Micronucleus (MN) formation is an endpoint which can be used to detect DNA damage resulting from clastogenic or aneugenic mechanisms. A sensitive and high throughput system to measure human blood for MN would have a myriad of biomonitoring applications. For example, such a system could provide information regarding the chromosome damaging activity of new drugs undergoing clinical trials, chemotherapy regimens, as well as accidental radiation or chemical exposures. A goal of the current application is to optimize methods developed during Phase 1 that allow for the flow cytometric measurement of MN in human reticulocytes (RETs0. Experiments are planned to test whether MN-RET measurements represent a sensitive indicator of cytogenetic damage. To achieve this goal, our Phase II aims are to. 1. COMPLETE optimization of cell staining and handling procedures. 2. DEVELOP biological standards to aid the calibration of flow cytometer parameters. 3. MEASURE the time-course and magnitude of MN-RET induction in radiotherapy and radio-plus chemo-therapy patients before and during the course of treatment. 4. MEASURE the time-course and magnitude of MN-RET induction in radiotherapy and radio-plus chemo-therapy patients before and during the course of treatment. 5. COLLABORATE with researchers in an environmental biomonitoring study of Chernobyl liquidators, clean-up workers and settlers. PROPOSED COMMERCIAL APPLICATIONS: The successful completion of this research project will enable Litron Laboratories to become an expert facility, capable of performing cytogenetic measurements on human blood samples on a fee-for-fee service basis. Additionally, by developing the necessary reagents into kit format, it will be possible to make this technology available to other laboratories having access to a single-laser flow cytometer. This technologically innovative technique has the potential to become an important clinical tool for measuring DNA damage carried by environmental exposures: radiation and/or chemotherapeutic therapies, aging and a myriad of other possible sources.