Bhas 42细胞转化试验高通量检测方法的建立
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摘要
目的建立基于96孔板的Bhas 42细胞转化试验高通量检测方法,并探讨此方法用于药物非临床阶段体外致癌性评价的前景。方法建立Bhas 42细胞转化试验96孔板高通量检测方法,使用已知阳性诱癌物苯并芘(Benzoapyrene,BaP)和环磷酰胺(Cyclophosphamide,CP)为对照品,比较转化灶人工计数法和过氧化氢高通量96孔板法对结果判定的影响。进行试验的细胞在接种后第19天以一定浓度的过氧化氢处理细胞24 h。24 h后,洗去含过氧化氢的培养基,加入新鲜培养基并加入CCK-8染色孵育2～4 h后测定450 nm波长吸光度以确定细胞转化率。同时,转化灶人工计数法细胞在第21天经甲醇固定,吉姆萨染液染色后计数每孔克隆数,对比两种方法的差异性。结果转化灶人工计数法中,与阴性对照组比较,BaP和CP在启动试验中均被评价为阳性;过氧化氢高通量法得到与转化灶人工计数法相同的结果。结论成功建立了基于96孔板的Bhas 42细胞转化试验的过氧化氢高通量检测方法,该方法相比于传统方法可简单、快速地检测化合物诱导Bhas42细胞的转化效率,并提高了结果评判的客观性。
Objective To establish a high throughput assay for Bhas 42 cell transformation assay based on 96-well plate, and to explore the prospect of this assay in evaluating the carcinogenicity of drugs in non-clinical phase in vitro.Methods A 96-well plate high-throughput assay for Bhas 42 cell transformation assay was established. Benzoapyrene(BaP) and Cyclophosphamide(CP)were used as positive control substances to compare the effects of the manual counting method and 96-well plate high-throughput assay on the results. The Bhas 42 cells were treated with hydrogen peroxide on nineteenth days after inoculation for 24 h. After 24 hours, the medium containing hydrogen peroxide was washed off, fresh medium with CCK-8 was added and incubate for 2—4 hours. The cell transformation rate was determined by measuring 450 nm wavelength absorbance. At the same time, the cells were fixed by methanol on the 21 st day and stained by Giemsa solution. The correlation between the two methods was statistically analyzed and compared.Results Compared with the negative control, both BaP and CP were positive in the initiation assay, and the results of high-throughput cell transformation assay were the same as manual counting cell transformation assay.Conclusion A 96-well plate-based high-throughput method for the detection of Bhas 42 cell transformation was successfully established. Compared with the traditional method, this method is easily and quickly, and improves the objectivity of the result evaluation.
Objective To establish a high throughput assay for Bhas 42 cell transformation assay based on 96-well plate, and to explore the prospect of this assay in evaluating the carcinogenicity of drugs in non-clinical phase in vitro.Methods A 96-well plate high-throughput assay for Bhas 42 cell transformation assay was established. Benzoapyrene(BaP) and Cyclophosphamide(CP)were used as positive control substances to compare the effects of the manual counting method and 96-well plate high-throughput assay on the results. The Bhas 42 cells were treated with hydrogen peroxide on nineteenth days after inoculation for 24 h. After 24 hours, the medium containing hydrogen peroxide was washed off, fresh medium with CCK-8 was added and incubate for 2—4 hours. The cell transformation rate was determined by measuring 450 nm wavelength absorbance. At the same time, the cells were fixed by methanol on the 21 st day and stained by Giemsa solution. The correlation between the two methods was statistically analyzed and compared.Results Compared with the negative control, both BaP and CP were positive in the initiation assay, and the results of high-throughput cell transformation assay were the same as manual counting cell transformation assay.Conclusion A 96-well plate-based high-throughput method for the detection of Bhas 42 cell transformation was successfully established. Compared with the traditional method, this method is easily and quickly, and improves the objectivity of the result evaluation.
引文
[1]王颖,蒲江,齐乃松,等. Bhas 42细胞转化试验高通量检测方法的建立及应用[J].癌变·畸变·突变, 2015,27(4):288-293.
[2]庞雅琴,陈雯.体外细胞转化试验研究进展[J].癌变·畸变·突变, 2007, 19(6):511-514..
[3]王颖,王雪,李波.两阶段细胞转化试验研究进展[J].中国新药杂志, 2013(10):1133-1136..
[4] Fontana C, Kirsch A, Seidel C, et al. In vitro cell transformation induced by synthetic amorphous silica nanoparticles[J]. Mutat Res, 2017, 823:22-27..
[5]王颖. Bhas42细胞转化试验方法学及其应用研究[D].北京:中国食品药品检定研究院, 2013.
[6] SASAKI K. A method for selecting mammal transformed cells:EP20090169631[P/OL]. 2009-09-07[2011-08-17].http://www.freepatentsonline.com/EP2163895B1.html.
[7] Sakai A, Sasaki K, Muramatsu D, et al. A Bhas 42 cell transformation assay on 98 chemicals:the characteristics and performance for the prediction of chemical carcinogenicity.[J]. Mutation Research, 2010, 702(1):100-122.
[8] Sakai A, Sasaki K, Hayashi K, et al. An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity[J].Mutation Research/genetic Toxicology&Environmental Mutagenesis, 2011, 725(1-2):57-77.
[9] Corvi R, Aardema M J, Gribaldo L, et al. ECVAM prevalidation study on in vitro cell transformation assays:general outline and conclusions of the study[J]. Mutation Research-Genetic Toxicology and Environmental Mutagenesis, 2012, 744(1):12-19.
[10] Sasaki, Kiyoshi c/o Food and Drug Safety Center. A method for selecting mammal transformed cells:EP2163895[P]. 2011.
[2]庞雅琴,陈雯.体外细胞转化试验研究进展[J].癌变·畸变·突变, 2007, 19(6):511-514..
[3]王颖,王雪,李波.两阶段细胞转化试验研究进展[J].中国新药杂志, 2013(10):1133-1136..
[4] Fontana C, Kirsch A, Seidel C, et al. In vitro cell transformation induced by synthetic amorphous silica nanoparticles[J]. Mutat Res, 2017, 823:22-27..
[5]王颖. Bhas42细胞转化试验方法学及其应用研究[D].北京:中国食品药品检定研究院, 2013.
[6] SASAKI K. A method for selecting mammal transformed cells:EP20090169631[P/OL]. 2009-09-07[2011-08-17].http://www.freepatentsonline.com/EP2163895B1.html.
[7] Sakai A, Sasaki K, Muramatsu D, et al. A Bhas 42 cell transformation assay on 98 chemicals:the characteristics and performance for the prediction of chemical carcinogenicity.[J]. Mutation Research, 2010, 702(1):100-122.
[8] Sakai A, Sasaki K, Hayashi K, et al. An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity[J].Mutation Research/genetic Toxicology&Environmental Mutagenesis, 2011, 725(1-2):57-77.
[9] Corvi R, Aardema M J, Gribaldo L, et al. ECVAM prevalidation study on in vitro cell transformation assays:general outline and conclusions of the study[J]. Mutation Research-Genetic Toxicology and Environmental Mutagenesis, 2012, 744(1):12-19.
[10] Sasaki, Kiyoshi c/o Food and Drug Safety Center. A method for selecting mammal transformed cells:EP2163895[P]. 2011.