大鼠体内碱性彗星试验方法的建立和验证  

Establishment and verification of in vivo alkaline comet assay in rats

作　　者：梅阿宁 傅鹏 张建军[2] MEI Aning;FU Peng;ZHANG Jianjun(School of Pharmacy,Anhui Medical University,Hefei 230032,China;Tianjin Institute of Pharmaceutical Research New Drug Evaluation Co.,Ltd.,Tianjin 300301,China)

机构地区：[1]安徽医科大学药学院,安徽合肥230032 [2]天津药物研究院新药评价有限公司,天津300301

出　　处：《药物评价研究》2021年第6期1253-1258,共6页Drug Evaluation Research

摘　　要：目的对体内碱性彗星试验的主要影响因素琼脂糖浓度、解旋时间和电泳时间进行探讨,建立大鼠体内碱性彗星试验方法,并用阳性对照药甲磺酸乙酯进行验证实验。方法肝细胞来源于健康雄性SD大鼠,制备3层单细胞凝胶玻片。第1层琼脂糖凝胶提前1天铺,选用3种不同浓度(1.00%、0.75%、0.50%)的正常熔点琼脂糖,选用3种不同终浓度(0.91%、0.68%、0.45%)的低熔点琼脂糖和单细胞混悬液铺第2层,第3层低熔点琼脂糖浓度与第2层相同。玻片裂解过夜后,4℃下分别避光解旋4种不同时间(20、30、40、60 min),在2~10℃新鲜电泳液(pH>13)中以有效电压0.7 V/cm分别电泳3种不同时间(20、30、40 min),中和并用无水乙醇脱水后干燥保存。染色后在荧光显微镜下拍照并保存,用CASP软件对细胞图像分析,以细胞尾DNA百分比衡量细胞的DNA损伤程度。将10只健康雄性SD大鼠随机分为溶媒对照组和阳性对照组,分别给生理盐水和甲磺酸乙酯(200 mg/kg),ig给药2次,间隔21 h,最后1次给药后3 h收集肝组织制备单细胞悬液,按照已建立的实验方法检测甲磺酸乙酯的遗传毒性。结果第1层使用0.75%的琼脂糖溶液铺片合适,使用终浓度为0.68%的低熔点琼脂糖和单细胞混悬液铺第2层琼脂糖凝胶时,铺片合适,分别选择解旋20 min和电泳20 min作为解旋时间和电泳时间。经方法学检测,甲磺酸乙酯遗传毒性结果为阳性。结论成功地建立了大鼠体内碱性彗星试验方法并验证成功,有利于彗星试验在新药安全评价中发挥更大的作用。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 were given physiological saline and EMS(200 mg/kg) respectively, and were administered by gavage twice at an interval of 21 hours. The liver tissues were collected three hours after the last administration and prepare a single cell suspension to detect the 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

关 键 词：体内碱性彗星试验 肝细胞 影响因素 甲磺酸乙酯 琼脂糖浓度 解旋时间 电泳时间 

分 类 号：R965.1[医药卫生—药理学]