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目的用放线菌素D(actinomycin D,ACTD)处理V79细胞后,获取处理后不同时段去细胞培养液(conditioned medium,CM)培养细胞,观察ACTD对靶细胞和旁观者细胞的细胞活力和凋亡的影响,研究ACTD能否诱导旁观者效应的发生,为研究化学物所致的旁观者效应及其机制提供实验依据。方法用不同剂量ACTD(0、0.2、0.4、0.8、1.6、3.2和6.4μmol/L)处理V79靶细胞1和4 h;选择3.2μmol/L ACTD作用1 h开始计时,分别在4、8、12和24 h取靶细胞去细胞培养液(CM),培养正常细胞24 h后,进行旁观者效应的观察。MTT法测定细胞活力;流式细胞仪检测细胞凋亡、坏死率。结果随着ACTD剂量的增加,靶细胞的活力下降,存在明显的剂量-效应关系(P<0.05)。3.2μmol/L作用1 h细胞活力接近50%且稳定。凋亡率随剂量增加而增加,而作用4 h各剂量组凋亡率下降坏死率升高,考虑到足够高的凋亡率又避免损失过多的旁观者因子,故选择3.2μmol/L作用1 h作为处理因素来获取CM。旁观者细胞存活率随着时段的延后增加而凋亡率逐渐下降(P<0.05),在24 h时段均有一个反弹,但均以4 h CM诱导的旁观者效应最强。旁观者细胞主要死亡类型是凋亡。结论 ACTD可以诱导旁观者效应的发生,4 h无细胞培养液(CM)诱导旁观者细胞损伤的作用最强。旁观者细胞死亡以凋亡为主。
Objective To investigate the effect of ACTD on the viability and apoptosis of target cells and bystander cells after V79 cells were treated with actinomycin D (ACTD) and cultured in conditioned media (CM) To study whether ACTD can induce the bystander effect and provide experimental evidence for studying the bystander effect and its mechanism caused by chemicals. Methods V79 target cells were treated with different doses of ACTD (0,0.2,0.4,0.8,1.6,3.2 and 6.4μmol / L) for 1 and 4 h, respectively, and 3.2 μmol / L ACTD for 1 h, The target cell decellularization (CM) cells were harvested at 12 and 24 h, and the normal cells were cultured for 24 h. The bystander effect was observed. Cell viability was measured by MTT assay. Apoptosis and necrosis rates were detected by flow cytometry. Results With the increase of ACTD dose, the viability of target cells decreased and there was a significant dose-response relationship (P <0.05). 3.2μmol / L effect 1 h cell viability close to 50% and stable. Apoptosis rate increased with the increase of dose, and the apoptotic rate of each dose of 4 h after treatment decreased necrosis rate, taking into account the high enough rate of apoptosis and avoid excessive bystander factor, so choose 3.2μmol / L role 1 h as a handling factor to get CM. Bystander cell survival rate decreased gradually with time delay (P <0.05), and rebounded at 24 h, but the bystander effect induced by 4 h CM was the strongest. The main type of death bystander cells is apoptosis. Conclusion ACTD can induce the bystander effect, and cell-free culture medium (CM) induced the bystander cell injury at 4 h. Bystander cell death is mainly apoptosis.