放线菌素D诱导非洲绿猴肾细胞(Vero)凋亡模型的建立

Establishment of Apoptosis Model in Vero Cells Induced by Actinomycin D

用不同剂量(0,0.25,0.5,1.0,2.0,4.0 mg/L)的放线菌素D(actinomycin D,ActD)诱导Vero细胞凋亡不同时间(0,12,24,36,48,60 h)。MTT法和流式细胞术的结果显示,以未处理的细胞作为对照组,各剂量ActD处理Vero细胞不同时间后,细胞的活力均下降,凋亡率均升高,且呈剂量和时间依赖效应。当ActD浓度为1 mg/L、作用时间为36 h,细胞的存活率为(0.55±0.01),凋亡率为(34.83±1.13)%。Gimesa染色、Hoechst33258染色表明,经ActD(1 mg/L)诱导36 h后Vero细胞形态学发生明显改变,出现膜小泡和凋亡小体形成等凋亡细胞特征;Real-time PCR结果显示,诱导组Bax/Bcl-2 mRNA相对含量明显大于正常对照组,且差异有统计学意义;分光光度法结果表明,与正常对照组相比较,凋亡诱导组caspase-3和caspase-8活性明显升高且差异具有统计学意义,caspase-9活性与对照组相比较,差异无统计学意义。用1 mg/L ActD诱导Vero细胞36 h时,细胞存活率和凋亡率都很高,可作为ActD诱导Vero细胞的最佳剂量。在该剂量及作用时间下,caspase-3和caspase-8活性明显升高,表明ActD诱导Vero细胞凋亡是通过caspase-8相关的途径。该实验成功建立了ActD诱导非洲绿猴肾细胞凋亡模型,将有助于进一步探讨目的基因在Vero细胞凋亡作用的的分子机制。

Vero cells were treated with different doses （0, 0.25, 0.5, 1.0, 2.0, 4.0 mg/L, respectively） of ActD for different times （0, 12, 24, 36, 48, 60 h）. MTT assay showed that the viabilities of Vero cells treated with ActD for different period and different concentrations decreased significantly compared with the control cells （P〈0.05）, and flow cytometry assay showed that the apoptosis rates of Vero cells treated with ActD for different period and concentrations all increased significantly compared with the control cells （P〈0.05）, the viabilities and the apoptosis rate showed a dose and time dependent effect. When the concentration of ActD was 1 mg/L, and the reaction time was 36 h, the cell viability was （0.55±0.01） and the apoptosis rate was reach to （34.83±1.13）%. Gimesa staining, Hochest33258 staining showed that the morphology of the Vero cell apparently changed and the tvnicalcharacteristic of apoptotic cells, for instance, membrane vesicles and apoptotic body was appeared after treated by ActD （1.0 mg/L） for 36 h. Real-time PCR demenstratated that compared with untreated Vero cells, Bax was increased, Bcl-2 was decreased in Vero cells treated by ActD （1.0 mg/L） for 36 h. Spectrophotometry results showed that compared with the control group, the caspase-3 and caspase-8 activity of apoptosis-induced group was significantly higher and the difference was statistically significant; while the activity of caspase-9 has no statistically sig- nificant difference compared with the control group. The optimum condition of ActD for establishment an apoptosis model in Vero cells is 1.0 mg/L for 36 h. At this condition, both the cell viability and the apoptosis rates were high. Caspase-3 and caspase-8 activity was significantly higher when dealed with ActD （1.0 mg/L） for 36 h, indicating that ActD-induced the Vero cell apoptosis is in caspase-8-dependent way. In this study, ActD-induced apoptosis model in Vero cells was successfully established which will help to further evaluate the molecular mechanisms of the role of target gene in apoptosis in Vero cells.