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

癌变·畸变·突变 ›› 2019, Vol. 31 ›› Issue (3): 242-248.doi: 10.3969/j.issn.1004-616x.2019.03.013

基于TK6细胞的体外PIG-A基因突变检测方法的建立

李若婉, 周长慧, 黄鹏程, 常艳

中国医药工业研究总院国家上海新药安全评价研究中心, 上海益诺思生物技术股份有限公司, 上海 201203

收稿日期:2018-09-28 修回日期:2018-12-19 出版日期:2019-05-31 发布日期:2019-05-31
通讯作者: 常艳,E-mail:ychang@ncdser.com E-mail:ychang@ncdser.com
作者简介:李若婉,E-mail:1162907345@qq.com。
基金资助:
十二五重大专项（2015ZX09501004-002-006）；上海市科学技术委员会计划项目（15140901100）

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

LI Ruowan, ZHOU Changhui, HUANG Pengcheng, CHANG Yan

China State Institute of Pharmaceutical Industry National Shanghai Center for New Drug Safety Evaluation and Research, Shanghai Innostar Bio-tech Company Limited, Shanghai 201203, China

Received:2018-09-28 Revised:2018-12-19 Online:2019-05-31 Published:2019-05-31

摘要/Abstract

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、100 μmol/L的EMS和4、8、16 μmol/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。

关键词: PIG-A基因, TK6细胞, 遗传毒性, 基因突变检测

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

中图分类号:

Q355

李若婉, 周长慧, 黄鹏程, 常艳. 基于TK6细胞的体外PIG-A基因突变检测方法的建立[J]. 癌变·畸变·突变, 2019, 31(3): 242-248.

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.

参考文献

[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.
[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, FRANKLIN D,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, PHONETHEPSWATH S, 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.

基于TK6细胞的体外PIG-A基因突变检测方法的建立

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	杨玉, 黄雅理, 林飞, 汤龙. 抗肿瘤血清胸腺因子9肽的急性毒性和遗传毒性[J]. 癌变·畸变·突变, 2022, 34(1): 57-61.
[2]	杜秀明, 洪丽玲, 徐灵芝, 周庆云. 采用体外染色体畸变试验检测锐钛型纳米二氧化钛的遗传毒性[J]. 癌变·畸变·突变, 2022, 34(1): 67-71.
[3]	蔡绮, 高德煜, 史建峰, 于浩, 韩妍, 杨柳, 赵蕾, 王召旭. 以树鼩为微核试验新型动物模型的初步研究[J]. 癌变·畸变·突变, 2021, 33(5): 370-374,382.
[4]	敬海明, 李国君, 宁钧宇. 苯丙(a)蒽的毒性研究进展[J]. 癌变·畸变·突变, 2021, 33(2): 149-152.
[5]	周长慧, 韩天娇, 黄鹏程, 马璟, 常艳. 大鼠多器官彗星试验方法的验证研究[J]. 癌变·畸变·突变, 2020, 32(3): 233-237.
[6]	霍娇, 朱雪娇, 曾珠, 刘运杰, 陈锦瑶, 张立实. 定量遗传毒性风险评估研究进展[J]. 癌变·畸变·突变, 2020, 32(2): 155-158,161.
[7]	曹易懿, 刘维映, 尤馨悦, 奚晶, 陈瑞雪, 张新宇, 栾洋. 多溴联苯醚BDE-3、BDE-47和BDE-209的体外遗传毒性评价[J]. 癌变·畸变·突变, 2020, 32(1): 15-20,28.
[8]	王全凯, 谢广云, 马顺鹏, 郭浩然, 乌瀚宝栎尔, 宋佳阳, 许建宁. 甲基丙烯酸环氧丙酯的遗传毒性评价[J]. 癌变·畸变·突变, 2019, 31(6): 479-482.
[9]	陈高峰, 王亚楠, 王丹, 毛志慧, 黄芝瑛, 文海若, 王雪. Pig-a基因突变试验研究进展[J]. 癌变·畸变·突变, 2019, 31(6): 492-497.
[10]	唐娇, 杨颖, 李庆, 赵敏, 杨杏芬. 利用中国仓鼠肺细胞体外微核试验评价三七提取液的遗传毒性[J]. 癌变·畸变·突变, 2019, 31(5): 397-400.
[11]	王亚楠, 文海若, 王雪. 遗传毒性基因突变评价方法的研究进展[J]. 癌变·畸变·突变, 2019, 31(5): 406-411.
[12]	文海若, 邵安良, 陈亮, 淡墨, 胡燕平, 王雪, 徐丽明. 适合纳米材料遗传毒性评价方法的选择[J]. 癌变·畸变·突变, 2018, 30(4): 326-331.
[13]	王丹, 耿兴超, 李波, 王雪, 文海若. 体外细胞转化试验的研究与应用进展[J]. 癌变·畸变·突变, 2018, 30(4): 310-314.
[14]	夏莹, 付少华, 张寅静, 王薇. 农药哌虫啶的遗传毒性试验[J]. 癌变·畸变·突变, 2018, 30(2): 136-139.
[15]	曹易懿, 奚晶, 唐伟锋, 尤馨悦, 刘维映, 栾洋. 三氯生的体外遗传毒性评价[J]. 癌变·畸变·突变, 2018, 30(1): 71-75.