放线菌素D诱导V79细胞凋亡模型的建立

Establishment of Apoptosis Model in V79 Cells Induced by Actinomycin D

目的确定RNA合成抑制剂——放线菌素D(actinomycin D,ACTD)诱发V79细胞凋亡的最适作用浓度和时间,建立细胞凋亡模型。方法采用不同剂量(0～8.0 mg/L)的ACTD染毒V79细胞不同时间(0.5～4 h)。采用MTT法检测细胞活力;采用流式细胞仪法分析细胞凋亡和坏死率。结果各剂量ACTD染毒不同时间后V79细胞活力均下降(P<0.05)。与染毒0.5 h比较,在染毒1 h后仅0.25、1、2、8 mg/L ACTD染毒V79细胞活力下降,在染毒2 h后0.25、1、2、4、8 mg/L ACTD染毒V79细胞活力下降(P<0.05),在染毒4 h后各剂量ACTD染毒V79细胞活力均下降(P<0.05)。各剂量ACTD染毒不同时间后V79细胞凋亡率均升高(P<0.05);与染毒0.5 h比较,各剂量ACTD染毒1 h以及0.25、0.5、1、2 mg/L ACTD染毒2、4 h后V79细胞凋亡率均升高(P<0.05),4 mg/L ACTD染毒4 h后V79细胞凋亡率下降(P<0.05)。与对照组比较,0.25、2mg/L ACTD染毒0.5 h,1、2、4 mg/L ACTD染毒2 h以及各剂量ACTD染毒4 h后V79细胞坏死率均升高(P<0.05);与染毒0.5 h比较,2 mg/L ACTD染毒1 h后V79细胞坏死率下降(P<0.05),1、4 mg/L ACTD染毒2 h以及0.5、1、2、4 mg/L ACTD染毒4 h后V79细胞坏死率均升高(P>0.05)。结论 ACTD诱发V79细胞凋亡的最适剂量为4 mg/L,最佳作用时间为1 h。

Objective To determine the optimum dose and duration of actinomycin D （ACTD, a RNA synthesis inhibitor） treatment to establish a model of apoptosis in V79 cells in order to investigate bystander effect. Methods V79 cells were treated with different doses （0,0.25,0.5,1.0,2.0,4.0 and 8.0mg/L, respectively） of ACTD for different duration （0.5,1,2 and 4 h） to detect cell viability, apoptosis and necrosis rate. Cell viability was estimated by M3T method. Apoptosis and necrosis rate of V79 cells was evaluated by flow cytometry assay. Results Cell viabilities of V79 cells treated with ACTD for different period decreased significantly compared with the control cells （P〈0.05）. Compared with 0.5 h, cell viabilities of V79 cells treated with ACTD for one hour decreased significantly at the doses of 0.25, 1, 2, 8 mg/L ACTD （P〈0.05）; while cell viabilities treated for 2 h decreased significantly at the doses of 0.25, 1, 2, 4, 8 mg/L ACTD （P〈0.05）; whereas cell viabilities treated for 4 h decreased significantly at all doses of ACTD （P〈0.05）. Apoptosis rates of V79 cells treated with ACTD for different period all increased significantly compared with the control cells （P〈0.05）. Compared with 0.5 h, apoptosis rates of V79 cells treated with ACTD for one hour at all doses and those for 2 h as well as 4 h at the doses of 0.25, 0.5, 1, 2 mg/L ACTD increased significantly （P〈0.05）. Also, 4 mg/L ACTD-treated cells apoptosis rates increased significantly when V79 cells treated for 4 h （P〈0.05）. Necrosis rates of V79 cells treated with ACTD for 0.5 h at the doses of 0.25,2 mg/L and treated with ACTD for 2 h at the doses of 1,2,4 mg/L and treated with ACTD for 0.5 h at all doses increased significantly compared with control cells （P〈 0.05）. Compared with 0.5 h, necrosis rates of V79 cells treated with ACTD for one hour at the doses of 2 mg/L decreased significantly（P〈0.05）;necrosis rates of V79 cells treated with ACTD 2 h at the doses of 1,4 mg/L and treated with ACTD for 4 h at the doses of 0.5, 1, 2,4 mg/L increased significantly （P〈0.05）. Conclusion The optimum condition of ACTD for establishment an apoptosis model in V79 cells is 4.0 mg/L for one hour.