流式细胞术检测体外微核方法的建立

目的： 建立96孔板流式细胞术体外微核自动化检测的方法，并探讨其用于药物早期遗传毒性筛选和遗传毒性评价的可能性。方法：试验分为+S9短时处理组(4 h)和-S9持续处理组(24 h)，分别选择3个不同浓度的环磷酰胺和丝裂霉素C处理CHO-K1细胞，24 h后收获细胞。采用EMA和SYTOX Green双色标记，流式细胞仪分析96孔板的微核率，并与常规标准平皿培养、细胞分裂阻滞法的双核微核结果进行比较。结果：在有或无S9处理条件下，不同浓度的环磷酰胺、丝裂霉素C诱导产生的微核率较溶剂对照组均显著增加(P均<0.05)，剂量-效应关系明显。两种微核检测方法的Spearman相关系数(rs)为1.000。结论：流式细胞术检测环磷酰胺、丝裂霉素C作用于CHO-K1细胞的微核试验结果均为阳性，与文献报道一致，因而本试验初步建立了流式细胞术体外微核检测方法。流式细胞术检测微核的方法与人工阅片的方法相关性好，提示该方法用于化合物早期遗传毒性筛选和遗传毒性评价具有良好的前景。

Establishment of flow cytometric in micronucleus assay in vitro

OBJECTIVE:Establish the flow cytometric 96-well microplate-basedin vitro micronucleus assay in CHO-K1 cells,and explore the possibility of this method for early genetic toxicity screening during drug discovery. MEHTODS:The test included treatment with and without metabolic activation. For the treatment with metabolic activation,CHO-K1 cells were treated with three different concentrations of cyclophosphamide in the S9 mixmedium for 4 h,then incubated with S9-free fresh medium for 20 h. For the treatment without metabolic activation,cells were incubated with three different concentrations of mitomycin C continuously for 24 h. In all cases,after a total of 24 h since initiation of the treatment,cells were processed for microscopic scoring or flow cytometric MN analysis. A flow cytometric method for scoring MN used EMA and SYTOX Green to label the cells in 96-well microplate,and then compared with cytokinesis-block micronucleus assay in cell culture disks based on microscopy.RESULTS:Mitomycin C and cyclophosphamide at different concerntrations caused statistically significant and dose-dependent increasess in micronucleus assay . Non-parametric Spearman's coefficients (rs) is 1.000.CONCLUSION:Similar to literature published,mitomycin C and cyclophosphamide induced positive results in flow cytometric based in vitro micronucleus assay. So the method of flow cytometric 96-well microplate-based in vitro micronucleus assay in CHO-K1 cells was established. The concordance between microscopic scoring and flow cytometricwas good,therefore this method is promising for screening and evaluating genetic toxicity of chemicals.