基于TK6细胞的体外PIG-A基因突变检测方法的建立

doi:10.3969/j.issn.1004-616x.2019.03.013

Abstract

Abstract: 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.

Key words: PIG-A gene, TK6 cell, genetic toxicity, gene mutation assay

CLC Number:

Q355

LI Ruowan, ZHOU Changhui, HUANG Pengcheng, CHANG Yan. Establishment of an in vitro PIG-A gene mutation assay based on TK6 cells for genotoxicity tests[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2019, 31(3): 242-248.

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Establishment of an in vitro PIG-A gene mutation assay based on TK6 cells for genotoxicity tests

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