优化胞质分裂阻滞实验方案用于核质桥分析的可行性

The optimized cytokinesis-block assay for radiation-induced nucleoplasmic bridge

目的探索优化胞质分裂阻滞实验方案用于辐射诱导核质桥分析的可行性,为以核质桥为指标进行生物剂量估算提供科学依据。方法用2 Gy ^(60)Coγ射线(剂量率为1 Gy/min)照射人离体外周血(设0 Gy对照),照射后28 h在细胞样品中加入终浓度为6μg/ml的松胞素B,培养48、56、68和72 h后收获;或照射后加入终浓度为0.6、1、2、6、10μg/ml的松胞素B,培养68 h后收获。应用胞质分裂阻滞法进行标本制备,分析单核细胞、双核细胞、多核细胞的比例,以及辐射诱导核质桥率及微核率。结果对不同细胞培养时间,随细胞培养时间的增加,0和2 Gy核分裂指数和双核细胞比例具有升高的趋势;2 Gy核质桥率无明显变化规律(0.0230~0.0330/细胞),差异无统计学意义(P>0.05),微核率差异亦无统计学意义(P>0.05)。对不同浓度松胞素B处理组,随松胞素B浓度的增加,0和2 Gy时核分裂指数和双核细胞比例有所升高;2 Gy核质桥率无明显变化规律(0.0230~0.0470/细胞),差异无统计学意义(P>0.05);与6μg/ml组相比,10μg/ml组微核率显著降低(U=2.74,P<0.01)。结论不同细胞培养时间组和不同松胞素B浓度组的辐射诱导核质桥率差异无统计学意义。适当缩短细胞培养时间可提前得到核质桥分析结果;培养开始加入松胞素B可简化实验步骤,但可供分析的细胞数过少,用于剂量估算的可行性尚需进一步研究。

Objective To explore the feasibility of the optimized cytokinesis-block(CB)assay on radiation-induced nucleoplasmic bridge(NPB),and to provide a scientific basis for the application of NPB in biological dose estimation.Methods Human peripheral blood in vitro was irradiated with 2 Gy 60Coγ-rays at a dose rate of 1 Gy/min(0 Gy control group).According to the culture time after irradiation,blood samples were divided into group 48,56,68 and 72 h.Cytochalasin-B(Cyt-B)with a concentration of 6μg/ml was added into the samples at 28 h and harvested at 48,56,68 and 72 h after irradiation,respectively.On the other hand,the blood samples were treated with different concentration of Cyt-B i.e.,0.6,1,2,6 and 10μg/ml at the beginning of culture(0 h)and harvested at 68 h after irradiation.The proportion of mononucleated,binucleated and multinucleated cells,radiation-induced NPB and micronucleus(MN)frequencies were analyzed.Results The nuclear division index(NDI)and proportion of binucleated cells at 2 Gy and 0 Gy had tendency of increasing with cell culture time.NPB frequencies(0.0230-0.0330/cell)and MN frequencies had no significantly difference(P>0.05).With the increase of Cyt-B concentration,NDI and the proportion of binucleated cells in group 2 Gy and 0 Gy also increased,but NPB frequencies(0.0230-0.0470/cell)had no significant difference(P>0.05).MN frequencies of group 10μg/ml were significantly lower than that of group 6μg/ml(U=2.74,P<0.01).Conclusions Cell culture time and Cyt-B concentration had no significant influence on radiation-induced NPB frequencies,suggesting that NPB could be obtained by appropriately reducing cell culture time and Cyt-B could be added into blood samples at the beginning of culture.But this protocol reduced the number of cells for further analysis,and thus its feasibility for dose estimation still need to be studied.