多细胞系胞质分裂阻滞微核细胞组学试验法的建立与应用

doi:10.3969/j.issn.1004-616x.2015.04.010

癌变·畸变·突变 ›› 2015, Vol. 27 ›› Issue (4): 304-308.doi: 10.3969/j.issn.1004-616x.2015.04.010

多细胞系胞质分裂阻滞微核细胞组学试验法的建立与应用

文海若, 淡墨, 齐乃松, 耿兴超, 王雪

中国食品药品检定研究院国家药物安全评价监测中心, 药物非临床安全评价研究北京市重点实验室, 北京 100176

收稿日期:2014-11-19 修回日期:2015-04-17 出版日期:2015-07-30 发布日期:2015-07-30
作者简介:文海若,E-mail:wenhairuo@nifdc.org.cn。
基金资助:
国家“重大新药创制”科技重大专项“药物安全评价技术平台”(2012ZX09302001)

Cytokinesis-block micronucleus cytomic test establishment in multiple mammalian cell lines

WEN Hairuo, DAN Mo, QI Naisong, GENG Xingchao, WANG Xue

Beijing Key Laboratory, National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, Beijing 100176, China

Received:2014-11-19 Revised:2015-04-17 Online:2015-07-30 Published:2015-07-30
Contact: 王雪,E-mail:xue_wang@nifdc.org.cn E-mail:xue_wang@nifdc.org.cn

摘要/Abstract

摘要： 目的: 采用不同组织来源的细胞系建立微核细胞组学,比较其遗传毒性生物标志物的敏感性,并研究雷公藤甲素(TPL)和甘草酸二铵(DG)对大鼠成纤维肺细胞(CHL)和犬肾小管上皮细胞系(MDCK)细胞微核率的影响。方法:首先建立方法,采用不同浓度丝裂霉素C(MMC)与CHL、L02、MDCK、Bhas42细胞作用6 h后给予细胞松弛素B(CytoB)继续培养约1.5个细胞倍增时间。收获细胞、制片、染色及镜检。计算每个剂量胞质分裂增殖指数(CBPI)、复制指数(RI)、坏死(Nec)及凋亡(Apop)细胞的千分率、双核细胞中微核(MN)、核芽(NBUD)和核质桥(NPB)出现的千分率。然后进行方法的验证,采用CHL细胞经不同浓度(1、10和100 μg/mL)DG预处理24 h后观察DG对上述细胞毒性和微核细胞组相关指标的影响。为进一步了解微核细胞组学对遗传毒性靶器官评估的效果,MDCK细胞经DG(10 μg/mL)预处理24 h后采用不同浓度(1和5 μg/mL)TPL染毒1 h,观察其对遗传毒性的影响。结果:MMC诱发的不同细胞系细胞毒性(CBPI、RI、Apop与Nec)均随MMC浓度升高呈升高趋势。各组细胞的MN、NBUD和NPB均出现不同程度的剂量相关性升高,但不同细胞系对MMC诱发的生物标志物峰值出现的敏感度不同。在验证实验中,与对照组相比,DG(10和100 μg/mL)对MMC(0.2 μg/mL)诱发的MN、NBUD和NPB升高有显著的抑制作用(P均<0.05)。TPL可诱发MDCK细胞NBUD和MN显著上升(P<0.05),且DG预处理可有效拮抗该效应(P<0.05)。结论:多细胞系胞质分裂阻滞微核细胞组学实验法可同时对多项遗传毒性指标进行评价。DG对CHL及MDCK细胞产生的染色体断裂有保护作用,在临床配伍中可发挥抗细胞DNA损伤的功效。

关键词: 胞质分裂阻滞, 微核, 微核细胞组学, 犬肾小管上皮细胞系, 雷公藤甲素, 甘草酸二铵

Abstract: OBJECTIVE: This study attempted to establish the cytokinesis-block micronucleus cytomic test and to compare the susceptibilities of genotoxic biomarkers in a range of mammalian cell lines. The effects of TPL (triptolide) and DG(diammonium glycyrrhizinate) on CHL and MDCK were also investigated. METHODS: Method development: Cells were treated with mitomycin C in different concentrations for 6 h,followed by a CytoB (cytochalasins B) treatment covering around 1.5 cell doubling time. The cells were subsequently harvested,prepared on slides,stained and examined microscopically. The average CBPI (cytokinesis block proliferation index) and RI (replicative index) of each dose,and the Nec(necrosis rate permillage), Apop(apoptosis rate permillage),as well as the MN (micronucleus rate permillage), NBUD(nuclear bud rate permillage),NPB(nucleoplasmic bridge rate permillage) in binucleated cells were estimated. Method application: CHL was pretreated with DG for 24 h before treated with MMC for 6 h to evaluate the effect of DG on the genotoxicity induced by MMC. MDCK was treated with TPL (1 or 5 μg/mL) for 1 h as the potential protective effect of DG was also examined. RESULTS: Cytokinesis-block micronucleus cytomic test was successfully established in CHL,MDCK,L02 and Bhas 42 cell lines. The cell toxicity(CBPI,RI,Apop and Nec)- related indexes increased along with increasing MMC concentrations. The MN,NBUD and NPB also exhibited dose-dependent rising trend induced by MMC,however,the sensitivity for the above biomarkers varied for different cell lines. DG(10 and 100 μg/mL) significantly reduced MMC (0.2 μg/mL)-induced increment on MN,NBUD and NPB(P<0.05). TPL(5 μg/mL) treatment produced elevations of MN and NBUD in MDCK(P<0.05),which could be attenuated by DG (10 μg/mL) significantly(P<0.05). CONCLUSION: Cytokinesis-block micronucleus cytomic test made it possible to effectively evaluate multiple genetic toxicity indexes within a single slide. Our data clearly demonstrated that the genotoxic effects triggered by TPL were predominately caused by the DNA breakage or loss,while DG showed potent protective effects against the chromosome breakage in both CHL and MDCK cell lines and was able to reduce the toxicities induced by other agent.

Key words: cytokinesis-block micronucleus assay, micronucleus cytomic, MDCK, triptolide, diammonium glycyrrhizinate

中图分类号:

R394.6
R99

文海若, 淡墨, 齐乃松, 耿兴超, 王雪. 多细胞系胞质分裂阻滞微核细胞组学试验法的建立与应用[J]. 癌变·畸变·突变, 2015, 27(4): 304-308.

WEN Hairuo, DAN Mo, QI Naisong, GENG Xingchao, WANG Xue. Cytokinesis-block micronucleus cytomic test establishment in multiple mammalian cell lines[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2015, 27(4): 304-308.

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多细胞系胞质分裂阻滞微核细胞组学试验法的建立与应用

Cytokinesis-block micronucleus cytomic test establishment in multiple mammalian cell lines

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