目的 對體內(nèi)堿性彗星試驗(yàn)的主要影響因素瓊脂糖濃度、解旋時間和電泳時間進(jìn)行探討，建立大鼠體內(nèi)堿性彗星試驗(yàn)方法，并用陽性對照藥甲磺酸乙酯進(jìn)行驗(yàn)證實(shí)驗(yàn)。方法 肝細(xì)胞來源于健康雄性SD大鼠，制備3層單細(xì)胞凝膠玻片。第1層瓊脂糖凝膠提前1天鋪，選用3種不同濃度（1.00%、0.75%、0.50%）的正常熔點(diǎn)瓊脂糖，選用3種不同終濃度（0.91%、0.68%、0.45%）的低熔點(diǎn)瓊脂糖和單細(xì)胞混懸液鋪第2層，第3層低熔點(diǎn)瓊脂糖濃度與第2層相同。玻片裂解過夜后，4℃下分別避光解旋4種不同時間（20、30、40、60 min），在2～10℃新鮮電泳液（pH>13）中以有效電壓0.7 V/cm分別電泳3種不同時間（20、30、40 min），中和并用無水乙醇脫水后干燥保存。染色后在熒光顯微鏡下拍照并保存，用CASP軟件對細(xì)胞圖像分析，以細(xì)胞尾DNA百分比衡量細(xì)胞的DNA損傷程度。將10只健康雄性SD大鼠隨機(jī)分為溶媒對照組和陽性對照組，分別給生理鹽水和甲磺酸乙酯（200 mg/kg），ig給藥2次，間隔21 h，最后1次給藥后3 h收集肝組織制備單細(xì)胞懸液，按照已建立的實(shí)驗(yàn)方法檢測甲磺酸乙酯的遺傳毒性。結(jié)果 第1層使用0.75%的瓊脂糖溶液鋪片合適，使用終濃度為0.68%的低熔點(diǎn)瓊脂糖和單細(xì)胞混懸液鋪第2層瓊脂糖凝膠時，鋪片合適，分別選擇解旋20 min和電泳20 min作為解旋時間和電泳時間。經(jīng)方法學(xué)檢測，甲磺酸乙酯遺傳毒性結(jié)果為陽性。結(jié)論 成功地建立了大鼠體內(nèi)堿性彗星試驗(yàn)方法并驗(yàn)證成功，有利于彗星試驗(yàn)在新藥安全評價中發(fā)揮更大的作用。

Objective The agarose concentration, unwinding time and electrophoresis time of the main influencing factors of the alkaline comet assay in vivo were discussed, and the alkaline comet assay method in rats was established, and the common positive control ethyl methanesulfonate (EMS) was used for verification experiment. Methods Hepatocytes were derived from healthy male SD rats, and three-layer single-cell gel slides were prepared. First layer of agarose gel was laid one day in advance, and three different concentrations of nomal melting point agarose (1.00%, 0.75%, and 0.5%) were used. Three different final concentrations of low melting point agarose and single cell suspension (0.91%, 0.68%, and 0.45%) were used as the second layer,and the third layer of low melting point agarose has the same concentration as the second layer. After the slides were lysed overnight,they were incubated in alkaline (pH > 13) electrophoresis buffer at 4 ℃ for four different time (20, 30, 40, 60 min), and then electrophoresed at an effective voltage of 0.7 V/cm for three different time (20, 30, and 40 min) at 2 ~ 10 ℃. After the electrophoresis was completed,the slides were dehydrated with abslute ethanol and then dried and stored. After staining with fluorescent dyes,taked pictures and saved them under a fluorescence microscope,analyzed with CASP, and measured the degree of DNA damage of the cells by the percentage of DNA in the cell tail. Ten healthy male SD rats were randomly divided into vehicle control group and positive control group. They genotoxicity of EMS according to the established experimental method. Results Use 0.75% agarose solution for the first layer to spread, and use low-melting agarose and single cell suspension with the final concentration of 0.68% to spread the second layer of agarose gel, which was suitable for spreading. Choose 20 min of unwinding and 20 min of electrophoresis as the unwinding time and electrophoresis time respectively. After methodological testing, the result of EMS genotoxicity was positive. Conclusion The laboratory has successfully established the in vivo alkaline comet assay method in rats and verified it successfully,which will help the comet assay play a greater role in the safety evaluation of new drugs.

R965.1