彗星实验方法的优化及影响因素的分析  

Optimization of comet experiment method and analysis of influencing factors

作　　者：范南英 张鹏 Fan Nanying;Zhang Peng(Dept of Biology,School of Basic Medicine,Center for Tissue Engineering and Stem Cell Research,Guizhou Province Key Laboratory of Regenerative Medicine,Guizhou Medical University,Guiyang 550025;National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique,Center for Tissue Engineering and Stem Cell Research,Guizhou Province Key Laboratory of Regenerative Medicine,Guizhou Medical University,Guiyang 550025)

机构地区：[1]贵州医科大学基础医学院生物学教研室,贵阳550025 [2]贵州医科大学细胞工程生物医药技术国家地方联合工程实验室,组织工程与干细胞实验中心,贵州省再生医学重点实验室,贵阳550025

出　　处：《安徽医科大学学报》2024年第2期219-223,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81960036);贵州省科技计划项目(编号:黔科合基础-ZK[2021]重点005);贵州省教育厅高校科技创新团队(编号:黔教技[2023]068)。

摘　　要：目的为了更有效地检测细胞DNA损伤,对彗星实验的影响因素进行优化并建立阳性对照。方法以传统碱性彗星实验为基础,对制胶方法、凝胶浓度、琼脂糖溶剂、裂解时间、细胞密度和电泳电压加以优化。以过氧化氢(H_(2)O_(2))作为DNA损伤诱导剂诱导HL-60细胞作为该实验的阳性对照。结果优化后的实验结果显示,制胶方法由三步法进步为两步法,以PBS为琼脂糖溶剂,0.8%正常熔点琼脂糖制备底层胶,0.7%低熔点琼脂糖与细胞悬液混合作为第二层胶,制胶效果好,操作更简单省时,较好地解决了脱胶问题。裂解1 h,1 V/cm电压电泳,获得了具有代表性的彗星图像,结果易于判读,能有效避免假阳性结果。并对不同浓度H_(2)O_(2)诱导的HL-60细胞DNA损伤情况进行比较,成功建立了彗星实验的阳性对照。结论与传统方法相比,优化后的彗星实验方法成本更低,更简单、快速、准确,重复性也更好,能够快速检测细胞中的DNA损伤。Objective To detect cell DNA damage more effectively by optimizing the influencing factors of Comet assay and establishing a positive control.Methods Based on the traditional alkaline comet assay,the gel preparation method,gel concentration,agarose solvent,lysis time,cell density and electrophoresis voltage were optimized.HL-60 cells were induced by hydrogen peroxide(H_(2)O_(2)) as a DNA damage inducer as a positive control.Results The optimized experimental results showed that the gel preparation method improved from three-step method to two-step method.PBS was used as agarose solvent,0.8% normal melting point agarose was used to prepare the bottom layer,and 0.7% low melting point agarose was mixed with cell suspension as the second layer.The gel preparation effect was better,the operation was simpler,the time was saved,and the degumming problem was better solved.After lysis for 1 h and electrophoresis at 1 V/cm voltage,representative comet images were obtained.The results were easy to interpret and could effectively avoid false positive results.The positive control of comet assay was successfully established by comparing the DNA damage of HL-60 cells induced by different concentrations of H_(2)O_(2).Conclusion Compared with the traditional method,the optimized comet assay method is simpler,faster,more accurate,lower cost and has better repeatability,which can quickly detect DNA damage in cells.

关 键 词：彗星实验 HL-60 DNA损伤模型 

分 类 号：Q2-33[生物学—细胞生物学]