基于TK6细胞的体外PIG-A基因突变检测方法的建立
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摘要
目的:建立基于人成淋巴TK6细胞的体外磷脂酰肌醇聚糖A类(PIG-A)基因突变试验方法,研究甲基磺酸乙酯(EMS)与苯并芘(B[a]P)诱导TK6细胞PIG-A基因突变的能力,并探讨该方法用于药物开发初期遗传毒性筛选和评价的前景。方法:通过免疫磁珠分离清除TK6细胞株中预先存在的GPI(-)细胞(即PIG-A基因突变细胞),然后连续40 d测定正常传代培养TK6细胞PIG-A基因的自发突变率。设不添加体外代谢活化系统长时处理组(24 h-S9)和添加体外代谢活化系统短时处理组(4 h+S9),并分别采用3个不同浓度50、75、100μmol/L的EMS和4、8、16μmol/L的B[a]P处理TK6细胞10 d,在第11天收获细胞。使用CD19、CD55、CD59抗体和核酸染料7-AAD对细胞进行标记,然后用流式细胞仪分析CD19阳性细胞中,CD55和CD59表达阴性的细胞频率。结果:经过免疫磁珠分离,TK6细胞中预先存在的PIG-A基因突变细胞频率降低至2.5×10~(-5)。连续测定40 d TK6细胞的PIG-A基因自发突变率,计算得到其自发突变率为5.04×10~(-7)个/d。与阴性对照组比较,不同浓度EMS和B[a]P诱导产生的PIG-A基因突变细胞比例均呈剂量依赖性增加,并超过阴性对照组的2倍以上。结论:本研究初步建立了基于TK6细胞的体外PIG-A基因突变检测方法。该方法成本较低,简便、快速,具有应用于药物早期遗传毒性筛选和遗传毒性评价的潜力。
OBJECTIVE: To develop an in vitro PIG-A mutation assay in human lymphoblastoid TK6 cells and to evaluate its capacity in determining mutagenicity of Ethyl methanesulfonate(EMS) and Benzo(a) pyrene(B[a]P). METHODS:In order to reach a high sensitivity of the assay,it was necessary to eliminate the pre-existing GPI(-) cells(PIG-A mutant cells) in TK6 cells by means of immunomagnetic beads,and then determine the spontaneous mutation rate of PIG-A gene in TK6 cells up to 40 days. In this study,TK6 cells were divided into two groups,long time treatment without metabolic activation system(24 h-S9 group)and short time treatment with metabolic activation system(4 h +S9 group),and were then treated with ethyl EMS(50,75,100 μmol/L) and B[a]P(4,8,16 μmol/L) of 3 concentration gradients,then determined the mutation rate of PIG-A gene on day 11. Cells were stained with APC Mouse-Anti-Human CD19 antibody,PE Mouse Anti-Human CD55 antibody, PE Mouse Anti-Human CD59 antibody and nucleic acid dye 7-AAD,then analyzed the negative frequencies of CD55 and CD59(ratio of CD55-CD59-/CD19~+ cells in CD19+cells).RESULTS: The level of preexisting mutant cells were reduced to lower than 75×10~(-6) after immunomagnetic beads separation. The spontaneous mutation rate for 40 days was 5.04×10~(-7) cells/day. In the 24 h-S9 group,different concentrations of EMS exerted a dose-dependent increase in the mutation rate of PIG-A gene compared with the negative control. In the 4 h +S9 group with 2-fold increase,B[a]P different concentrations resulted in a similar dose-dependent effect. CONCLUSION: The flow cytometric in vitro PIG-A mutation assay was developed in human lymphoblastoid TK6 cells. This assay has the advantages of the low cost,easy and fast procedure,which may be useful for early genotoxicity testing of chemicals and pharmaceuticals.
OBJECTIVE: To develop an in vitro PIG-A mutation assay in human lymphoblastoid TK6 cells and to evaluate its capacity in determining mutagenicity of Ethyl methanesulfonate(EMS) and Benzo(a) pyrene(B[a]P). METHODS:In order to reach a high sensitivity of the assay,it was necessary to eliminate the pre-existing GPI(-) cells(PIG-A mutant cells) in TK6 cells by means of immunomagnetic beads,and then determine the spontaneous mutation rate of PIG-A gene in TK6 cells up to 40 days. In this study,TK6 cells were divided into two groups,long time treatment without metabolic activation system(24 h-S9 group)and short time treatment with metabolic activation system(4 h +S9 group),and were then treated with ethyl EMS(50,75,100 μmol/L) and B[a]P(4,8,16 μmol/L) of 3 concentration gradients,then determined the mutation rate of PIG-A gene on day 11. Cells were stained with APC Mouse-Anti-Human CD19 antibody,PE Mouse Anti-Human CD55 antibody, PE Mouse Anti-Human CD59 antibody and nucleic acid dye 7-AAD,then analyzed the negative frequencies of CD55 and CD59(ratio of CD55-CD59-/CD19~+ cells in CD19+cells).RESULTS: The level of preexisting mutant cells were reduced to lower than 75×10~(-6) after immunomagnetic beads separation. The spontaneous mutation rate for 40 days was 5.04×10~(-7) cells/day. In the 24 h-S9 group,different concentrations of EMS exerted a dose-dependent increase in the mutation rate of PIG-A gene compared with the negative control. In the 4 h +S9 group with 2-fold increase,B[a]P different concentrations resulted in a similar dose-dependent effect. CONCLUSION: The flow cytometric in vitro PIG-A mutation assay was developed in human lymphoblastoid TK6 cells. This assay has the advantages of the low cost,easy and fast procedure,which may be useful for early genotoxicity testing of chemicals and pharmaceuticals.
引文
[1]MIURA D,DOBROVOLSKY V N,KASAHARA Y,et al.Development of an in vivo gene mutation assay using the endogenous Pig-A gene:I.Flow cytometric detection of CD59-negative peripheral red blood cells and CD48-negative spleen T-cells from the rat[J].Environ Mol Mutagen,2010,49(8):614-621.
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[5]KRUGER C T,HOFMANN M,HARTWIG A.The in vitro PIG-A gene mutation assay:mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol(GPI)status of TK6 cells[J].Arch Toxicol,2015,89(12):2429-2443.
[6]REES B J,TATE M,LYNCH A M,et al.Development of an in vitro PIG-A gene mutation assay in human cells[J].Mutagenesis,2017,32(2):283-297.
[7]NICKLAS J A,CARTER E W,ALBERTINI R J.Both PIGA and PIGL mutations cause GPI-a deficient isolates in the Tk6 cell line[J].Environ Mol Mutagen,2015,56(8):663-673.
[8]MCKINZIE P B,REVOLLO J R.Whole genome sequencing of mouse lymphoma L5178Y-3.7.2C(TK(+/-))reveals millions of mutations and genetic markers[J].Mutat Res Genet Toxicol Environ Mutagen,2017,814(1):1-6.
[9]DAVID R,TALBOT E,ALLEN B,et al.The development of an in vitro PIG-A assay in L5178Y cells[J].Arch Toxicol,2018,92(4):1609-1623.
[10]BEMIS J C,AVLASEVICH S L,LABASH C,et al.Glycosylphosphatidylinositol(GPI)anchored protein deficiency serves as a reliable reporter of PIG-A gene mutation:Support from an in vitro assay based on L5178Y/Tk(+/-)cells and the CD90.2 antigen[J].Environ Mol Mutagen,2018,59(1):18-29.
[11]WANG Y,REVOLLO J,MCKINZIE P,et al.Establishing a novel PIG-A gene mutation assay in L5178YTk(+/-)mouse lymphoma cells[J].Environ Mol Mutagen,2018,59(1):4-17.
[12]GLAAB W E,TINDALL K R.Mutation rate at the HPRT locus in human cancer cell lines with specific mismatch repair-gene defects[J].Carcinogenesis,1997,18(1):1.
[13]FOWLER P,SMITH R,SMITH K,et al.Reduction of misleading("false")positive results in mammalian cell genotoxicity assays.II.Importance of accurate toxicity measurement[J].Mutat Res,2012,747(1):104-117.
[14]FOWLER P,SMITH K,YOUNG J,et al.Reduction of misleading("false")positive results in mammalian cell genotoxicity assays.I.Choice of cell type[J].Mutat Res,2012,742(1/2):11-25.
[15]DERTINGER S D,BRYCE S M,PHONETHEPSWATHS,et al.When pigs fly:immunomagnetic separation facilitates rapid determination of PIG-A mutant frequency by flow cytometric analysis[J].Mutat Res,2011,721(2):163-170.
[2]KINOSHITA T.Biosynthesis and deficiencies of glycosylphosphatidylinositol[J].Proc Jpn Acad Ser B Phys Biol Sci,2014,90(4):130-143.
[3]DERTINGER S D,PHONETHEPSWATH S,FRANKLIND,et al.Integration of mutation and chromosomal damage endpoints into 28-day repeat dose toxicology studies[J].Toxicol Sci,2010,115(2):401-411.
[4]PHONETHEPSWATH S,BRYCE S M,BEMIS J C,et al.Erythrocyte-based PIG-A gene mutation assay:Demonstration of cross-species potential[J].Mutat Res Genet Toxicol Environ Mutagen,2008,657(2):122-126.
[5]KRUGER C T,HOFMANN M,HARTWIG A.The in vitro PIG-A gene mutation assay:mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol(GPI)status of TK6 cells[J].Arch Toxicol,2015,89(12):2429-2443.
[6]REES B J,TATE M,LYNCH A M,et al.Development of an in vitro PIG-A gene mutation assay in human cells[J].Mutagenesis,2017,32(2):283-297.
[7]NICKLAS J A,CARTER E W,ALBERTINI R J.Both PIGA and PIGL mutations cause GPI-a deficient isolates in the Tk6 cell line[J].Environ Mol Mutagen,2015,56(8):663-673.
[8]MCKINZIE P B,REVOLLO J R.Whole genome sequencing of mouse lymphoma L5178Y-3.7.2C(TK(+/-))reveals millions of mutations and genetic markers[J].Mutat Res Genet Toxicol Environ Mutagen,2017,814(1):1-6.
[9]DAVID R,TALBOT E,ALLEN B,et al.The development of an in vitro PIG-A assay in L5178Y cells[J].Arch Toxicol,2018,92(4):1609-1623.
[10]BEMIS J C,AVLASEVICH S L,LABASH C,et al.Glycosylphosphatidylinositol(GPI)anchored protein deficiency serves as a reliable reporter of PIG-A gene mutation:Support from an in vitro assay based on L5178Y/Tk(+/-)cells and the CD90.2 antigen[J].Environ Mol Mutagen,2018,59(1):18-29.
[11]WANG Y,REVOLLO J,MCKINZIE P,et al.Establishing a novel PIG-A gene mutation assay in L5178YTk(+/-)mouse lymphoma cells[J].Environ Mol Mutagen,2018,59(1):4-17.
[12]GLAAB W E,TINDALL K R.Mutation rate at the HPRT locus in human cancer cell lines with specific mismatch repair-gene defects[J].Carcinogenesis,1997,18(1):1.
[13]FOWLER P,SMITH R,SMITH K,et al.Reduction of misleading("false")positive results in mammalian cell genotoxicity assays.II.Importance of accurate toxicity measurement[J].Mutat Res,2012,747(1):104-117.
[14]FOWLER P,SMITH K,YOUNG J,et al.Reduction of misleading("false")positive results in mammalian cell genotoxicity assays.I.Choice of cell type[J].Mutat Res,2012,742(1/2):11-25.
[15]DERTINGER S D,BRYCE S M,PHONETHEPSWATHS,et al.When pigs fly:immunomagnetic separation facilitates rapid determination of PIG-A mutant frequency by flow cytometric analysis[J].Mutat Res,2011,721(2):163-170.