放线菌素D对V79靶细胞和旁观者细胞凋亡的影响  

Effect of actinomycin D on apoptosis of V79 target and bystander cells

作　　者：靳翠红[1] 巫生文[1] 安丽[1] 刘秋芳[1] 逯晓波[1] 蔡原[1] 

机构地区：[1]中国医科大学公共卫生学院毒理教研室,辽宁沈阳110001

出　　处：《毒理学杂志》2012年第2期88-91,共4页Journal of Toxicology

基　　金：国家自然基金项目资助(30471475)

摘　　要：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 treat V79 cells with actinomycin D（ACTD） to get different period conditioned media（CMS） and observe the effect of ACTD and CM on cell viability and apoptosis/necrosis rate of V79 cells to determine whether ACTD could induce bystander effect in order to provide the experimental data to explore the mechanism underlying bystander effect.Methods V79 cells were administrated with ACTD for 1 h and 4 h at the different doses（0,0.2,0.4,0.8,1.6,3.2 and 6.4 μmol/L,respectively）.And different period conditioned media were collected at 4,8,12 and 24 h after cells were treated with 3.2 μmol/L ACTD for 1h to culture bystander cells to observe the bystander effect.Cell viability was evaluated by MTT assay;Apoptosis or necrosis of V79 cells were evaluated by flow cytometry assay.Results Cell viability of V79 cells reduced in ACTD-treated groups in a dose-dependent manner（P0.05）,among which cell viability was close to 50 percent and stable when ACTD was at 3.2 μmol/L for 1h.Apoptosis rate increased with the dose of ACTD treated for 1h but decreased when treated for 4h.To avoid the loss of bystander factors we chose the condition（3.2 μmol/L ACTD treatment for 1 h） to harvest conditioned medium.Cell viability increased significantly and apoptosis rate decreased significantly in a time-dependent manner（P0.05） with a rebound in 24 h CM,among which the bystander effect in 4 h CM was the strongest.Death type of bystander cells mainly was apoptotic.Conclusion ACTD could induce bystander effect in V79 cells.4 h CM could induce the strongest bystander effect.The bystander cell died mostly through apoptosis.

关 键 词：放线菌素D 条件培养液 旁观者效应 

分 类 号：R730.53[医药卫生—肿瘤]