Measuring Cytogenetic Damage in Human Blood

人类通过各种来源暴露于遗传毒性物质。微核（MN）的形成是一个终点，它可以用来检测DNA损伤所造成的染色体断裂或非整倍体机制。一个灵敏的和高通量的系统来测量人的血液MN将有无数的生物监测应用。例如，这样的系统可以提供有关正在进行临床试验、化疗方案以及意外辐射或化学暴露的新药的染色体损伤活性的信息。本申请的目标是优化在第1阶段期间开发的允许流式细胞术测量人网织红细胞（RETs）中MN的方法。实验计划测试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.细胞染色和处理程序的复杂优化。2.生物标准品，以帮助校准流式细胞仪参数。3.在放疗和放化疗患者中，在治疗前和治疗过程中测量MN-RET诱导的时间过程和幅度。4.在放疗和放化疗患者中，在治疗前和治疗过程中测量MN-RET诱导的时间过程和幅度。5. 与研究人员合作，对切尔诺贝利清理者、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实验室成为一个专家机构，能够在收费服务的基础上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的形式，将有可能使这项技术可用于其他拥有单激光流式细胞仪的实验室。这种技术创新的技术有可能成为一个重要的临床工具，用于测量环境暴露所带来的DNA损伤：辐射和/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.