目的 建立大鼠肝細(xì)胞和外周血淋巴細(xì)胞的體內(nèi)彗星試驗(yàn)方法，并應(yīng)用該方法對遺傳毒性陽性物甲磺酸乙酯（EMS）和環(huán)磷酰胺（CPA）進(jìn)行檢測。方法 選擇SD雄性大鼠為實(shí)驗(yàn)動物，分別給予不同劑量EMS和CPA。給藥結(jié)束后，采集外周血樣本和摘取動物左側(cè)肝葉組織樣本，鋪制成單細(xì)胞凝膠玻片。玻片經(jīng)裂解、解旋、電泳、中和、脫水等步驟后，得到可觀察的實(shí)驗(yàn)標(biāo)本。染色標(biāo)本，并在40×熒光顯微鏡下觀察拍照，使用專業(yè)分析軟件對單細(xì)胞電泳圖像進(jìn)行分析和測定。結(jié)果 成功建立了基于大鼠肝細(xì)胞和外周血淋巴細(xì)胞的體內(nèi)彗星試驗(yàn)方法。經(jīng)該方法檢測，EMS結(jié)果為陽性，CPA結(jié)果為陰性，EMS適合作為本方法的陽性對照。結(jié)論 成功建立大鼠體內(nèi)彗星試驗(yàn)方法，并可應(yīng)用于遺傳毒性檢測。

Objective To establish the comet assay in vivo method used for rat liver and peripheral blood lymphocyte cells and detect the genotoxicity of ethyl methanesulfonate (EMS) and cyclophosphamide (CPA) Methods Sprague-Dawley (SD) male rats were treated with series doses of EMS and CPA. Peripheral blood sample and tissue in left lobe of liver were collected to prepare single cell gel suspension and pave the slides. The slides were processed with complex steps containing lysis, unwinding, electrophoresis, neutralizing, and dehydrating to get the scoring specimen. To dye the specimens, observe the comet cells under fluorescence microscope (40×), and then photograph. The cell images were analyzed with special software and determined by electrophoresis. Results EMS showed positive reaction while CPA was negative in two tissues. It was convinced that EMS was appropriate as positive reagent in this assay. Conclusion The method of rat comet assay in vivo is successfully established and it could be used to genotoxicity detection.

重大新藥創(chuàng)制科技重大專項(xiàng)（2008ZX09305-002）