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目的 建立大鼠肝细胞和外周血淋巴细胞的体内彗星试验方法 ,并应用该方法对遗传毒性阳性物甲磺酸乙酯(EMS)和环磷酰胺(CPA)进行检测。方法选择SD雄性大鼠为实验动物,分别给予不同剂量EMS和CPA。给药结束后,采集外周血样本和摘取动物左侧肝叶组织样本,铺制成单细胞凝胶玻片。玻片经裂解、解旋、电泳、中和、脱水等步骤后,得到可观察的实验标本。染色标本,并在40×荧光显微镜下观察拍照,使用专业分析软件对单细胞电泳图像进行分析和测定。结果成功建立了基于大鼠肝细胞和外周血淋巴细胞的体内彗星试验方法。经该方法检测,EMS结果为阳性,CPA结果为阴性,EMS适合作为本方法的阳性对照。结论成功建立大鼠体内彗星试验方法 ,并可应用于遗传毒性检测。
OBJECTIVE: To establish a method for in vivo comet assay of rat hepatocytes and peripheral blood lymphocytes, and to detect the genotoxicity positive metabolites ethyl methanesulfonate (EMS) and cyclophosphamide (CPA) using this method. Methods SD male rats were selected as experimental animals and given different doses of EMS and CPA respectively. After the administration, peripheral blood samples were taken and the animal’s left liver lobe tissue samples were taken and made into single-cell gel slides. After the slides were lysed, untwisted, electrophoresed, neutralized and dehydrated, the observable experimental specimens were obtained. Stained specimens, and observed under a 40 × fluorescence microscope to take pictures, the use of professional analysis software for single cell electrophoresis image analysis and determination. Results The in vivo comet assay based on rat hepatocytes and peripheral blood lymphocytes was successfully established. The method of detection, EMS results were positive, CPA results were negative, EMS suitable as a positive control of this method. Conclusion The method of comet assay in rat is successfully established and can be applied to the detection of genotoxicity.