三种细胞形态学分析方法在体外微核检测中的比较研究

Comparative study of three cell morphological analysis methods on in vitro micronucleus assay

【目的】探讨基于流式细胞术(FCM)和细胞高内涵筛选技术(HCS)的两种高通量体外微核试验方法应用于化学品遗传毒性评价的可行性。【方法】参考经济合作与发展组织(OECD)TG487推荐的程序方法,采用化学品遗传毒性评价的典型阳性剂环磷酰胺(CP)、丝裂霉素C(MMC)为受试物,CP剂量范围为5~20 mg/L,MMC剂量范围为0.25~1.0 mg/L,分别设3个剂量组,短时处理(4 h)体外培养的CHL细胞模型,分别建立基于FCM和HCS的高通量体外微核测试方法。平行采用传统胞质分裂阻断微核法,在加/不加代谢活化系统条件下,检测各剂量组细胞的微核形成情况,并分析微核细胞率。实验同步设置阴性对照组(无血清MEM培养液)。【结果】经常规人工显微镜阅片和高通量方法的FCM及HCS得到的CP、MMC体外微核试验结果均为阳性:经CP诱导产生的微核细胞率(CP浓度由低到高)依次为1.9%、7.6%、10.4%(人工阅片),2.8%、2.6%、7.8%(FCM),2.8%、6.2%、9.1%(HCS);经MMC诱导产生的微核细胞率(MMC浓度由低到高)依次为5.9%、11.4%、16.7%(人工阅片),3.2%、3.7%、5.1%(FCM),7.9%、10.1%、10.2%(HCS)。受试物各剂量组与阴性对照组相比,差异均有统计学意义(P<0.05),且呈现剂量-反应关系。【结论】在本试验条件下,流式细胞术、高内涵体外微核检测的方法与传统人工阅片的方法得到的检测结果均一致,提示体外微核高通量方法可以实现细胞微核形成的自动化检测,提高检测效率,可为将高通量体外微核测试方法纳入化学品遗传毒性评价相关标准提供数据支持。

[Objective]To investigate the feasibility of genotoxicity assessment for chemicals via flow cytometry(FCM)and high-content screening(HCS)based on high-throughput screening in vitro micronucleus assays.[Methods]In reference to the methodology of OECD TG487,the typical positive controls,cyclophosphamide(CP)and mitomycin C(MMC),were selected.And no serum MEM medium was treated as negative control.Dose range of CP was 5-20 mg/L and MMC was 0.25-1.0 mg/L.CHL cells were treated with three concentrations of each chemical for 4 h.High-throughput screening in vitro micronucleus assays based on FCM and HCS were established.The results of the frequency of micronuclei were compared to traditional cytokinesis blocking micronucleus assay in each group with or without metabolic activation.[Results]The frequencies of micronuclei induced by CP and MMC(ascending rank)were separately 1.9%,7.6%,10.4%and 5.9%,11.4%,16.7%,which were obtained by conventional microscopic scoring.The frequencies of micronuclei induced by CP and MMC(ascending rank)were separately 2.8%,2.6%,7.8%and 3.2%,3.7%,5.1%,which were obtained by flow cytometry screening.The frequencies of micronuclei induced by CP and MMC(ascending rank)were separately2.8%,6.2%,9.1%and 7.9%,10.1%,10.2%,which were obtained by high-content screening.Compared with negative controls,the differences of the results were statistically significant(P<0.05),and there was a dose-response relationship.[Conclusion]In this study,the results of high-throughput screening assays of FCM and HCS are in accordance to the results of traditional cytokinesis blocking micronucleus assay,indicating that high-throughput screening in vitro micronucleus assays could detect micronucleus formation automatically and improve the efficiency.Therefore,the method could provide data support for using high-throughput screening in vitro micronucleus assays into genotoxicity assessment of chemicals.